Clase 5

MódemopcionalPaso 1. Conmutador de posición Paso 3. Conectar la alimentaciónPaso 4. Comprobar el estadoGS208Conmutadores Gigabit GS205/GS208 de 5 y 8 puertosRouter opcional InternetEquipoServidorGuía de instalaciónEncendidoApagadoIndicador de alimentación Actividad (intermitente)Sin conexión (apagado)Conexión Ethernet Indicadores de puertosPaso 2. Conectar el equipoServicio técnicoGracias por elegir un producto NETGEAR.Una vez instalado el dispositivo, busque el número de serie en la etiqueta del producto y regístrelo en https:// .Deberá registrar su producto NETGEAR recomienda registrar su producto a través de la página web de NETGEAR. Podráencontrar actualizaciones del producto y asistencia técnica en .NETGEAR recomienda utilizar sólo los recursos de soporte oficiales NETGEAR.EspecificacionesDescripciónInterfaz de red Conector RJ-45 para 10BASE-T, 100BASE-TX o1000BASE-TCable de red Cable Ethernet categoría 5 (Cat 5) o superior PuertosGS205: 5GS208: 8Fuente de alimentación *****************Consumo de energía GS205: 3,56W GS208: 3,64WPesoGS205: 0,12 Kg (0,27 lbs)GS208: 0,19 Kg (0,43 lbs)Dimensiones:(anchura x profundidad x altura)GS205: 114 mm x 86 mm x 26 mm(4.49 pulg. x 3.39 pulg. x 1.02 pulg.)GS208: 152 mm x 93 mm x 26 mm(5.98 pulg. x 3.66 pulg. x 1.02 pulg.)Temperatura de funcionamiento 0°–40° C (32°–104° F)Humedad de funcionamiento Humedad relativa (sin condensación) del 10% al 90%CertificacionesMarca CE, CB, CE Clase B, C-Tick Clase BEnero de 2013Para la actual declaración de conformidad de la UE actual, visite /app/answers/detail/a_id/11621/.NETGEAR, el logotipo de NETGEAR y Connect with Innovation son marcascomerciales o marcas comerciales registradas de NETGEAR, Inc. o sus filiales en Estados Unidos y otros países. La información contenida en el documento puede sufrir modificaciones sin previo aviso. El resto de marcas y nombres de productos son marcas comerciales o marcas comerciales registradas de sus respectivos titulares.© 2013 NETGEAR, Inc. Todos los derechos reservados.Sólo para uso interior en todos los Estados miembros de la UE, estados de la AELE y Suiza.。

现西第二册Lección 1【一】语法过去分词做形容词的构成：第一变位动词去掉词尾ar加ado，第二、三变位动词去掉词尾er或ir加ido。

几个特殊变化动词：Hacer→hecho decir→dicho oír→oídoPoner→puesto escribir→escrito abrir→abiertoVolver→vuerto morir→muerto ver→vistoDespertar→despierto醒着的，机灵的Atender→atendido(接待的)或atento(专注的)要切记过去分词做形容词修饰名词时要有“性、数”的变化。

【二】Léxico词汇I.Quitar(se)A. tr.摘，去掉quitar una cosa de una lugar1.Vamos a quitar este mapa de la pared, que ya no sirve.我们要把这个地图从墙上取下来，它已经没用了。

2.Por favor,quiten todas esas cosas de la mesa.请把桌子上的所有东西都拿走。

3.Tuve que quitar muchas palabras del texto,porque no las comprendían los alumnos.我应该巴课文里的很多单词去掉了，因为学生们对它们都不太会。

B.prnl.脱下quitarse una cosa1.Quítate el abrigo.你把大衣脱了吧。

2.Se quitó los zapatos para probarse los que quería comprar.他脱了鞋子来试试他想买的（鞋子）。

C.tr.夺去，夺下quitar una cosa a uno madre le quitó el pan de la mano al niño y le dijo: Lávate las manos antes.母亲从孩子手里夺过面包并对他说：先洗洗你的手去。

《生化危机5》全宝物指南详解注意：1、宝物一共有50个，不规则的有15个，规则的有35个。

2、其中梨状的（Pear）、方形的（Square）、鸡蛋形的（Oval）、三角形的（Trilliant）、花朵形的（Brilliant）尖椭圆形的（Marquis）心形的（最高级宝石，英文名不固定），各有5个。

颜色排列分别是：黄色（Topaz）、红色（Ruby）、蓝色（Sapphire）、绿色（Emerald）、白色（Diamond），对照自己已经取得的宝石，很容易就可以知道还有哪些没拿到。

3、宝物拿到后可以马上卖掉，只要曾经拿到就可以解开成就。

下面按排列顺序详细解释下各个宝石的取得条件：金戒指（Gold Ring）：很多地方可以拿到，最开始1-1的时候干掉斧男可以掉一个。

象牙护符（Ivory Relief）：1-2被SM的金发美女，干掉后会掉一个。

亡灵新娘之项链（Dead Bride's Necklace）：5-2的传送带上可以捡到，6-1的火箭筒丧尸也会掉。

皇家项链（Royal Necklace）：5-3有很多火箭筒丧尸围着的控制室里的桌上。

珠宝手镯（Jewel Bangle）：黑胖子身上都有。

毒牙（Venom Fang）：干掉电锯会掉。

注意第一个电锯在Veteran难度以下不会掉，只会掉钥匙。

Veteran难度以上干掉后先掉钥匙，之后复活，再干掉也会掉一个。

古董座钟（Antique Clock）：1-2 打金发女之前，那个二楼门从里面锁住的小楼，从底层上去，在阳台的宝箱里。

银圣杯Chalice (Silver) ：3-1沼泽中央的小屋里。

金圣杯Chalice (Gold) ：5-3有铁吊桥和很多舔食者的场景，在控制室的储物柜里。

银图腾（Silver Idol）：3-1右侧岸边有两个需要协力才能取得的宝箱，一个里面是石板碎片，另一个里面就是宝物。

金图腾（Gold Idol）：4-2镜子谜题的最后一层，先让光线照到雕像上，在开启的房间里。

八年级英语下册语法填空单元练习题(含答案)一、八年级英语下册语法填空专项练习（含答案解析）1．阅读下面短文，在空白处填入一个适当的词，或填入括号中所给单词的正确形式。

It's autumn now. The weather is cool and comfortable. It's a good time for ________ （walk）. So, our teacher organized a climbing for us. We decided ________ （climb） the mountains in our city.We went there ________ bus and arrived there at 9 a.m. ________ we got off the bus, we started to climb the mountain. The mountain is high. We were so excited ________ we climbed fast at first. But after a while, some of us ________ （be） tired, especially for girls. They did not want to go on anymore. ________, the others encouraged（鼓励）and helped them. All of ________（we） climbed slowly so that no one was left behind. ________ about 11:00 a. m, we ________ （get） to the top of the mountain. We were tired but really happy.2．语法填空An artist painted many pictures of great beauty. But he found that he had not yet painted a "real" picture.He began his trip to look ________ the most beautiful thing in the world. ________the way, he met an aged priest（神父）who ________ （ask）him where he was going. "I don't know, "said the artist." I want ________ （paint）the most beautiful thing in the world. Perhaps you can direct me to it. ""How easy, "replied the priest." In any church, you will find it 'Faith'（信仰, 信任）is the most beautiful thing in the world. "The artist traveled on. ________ （late）, a young mother told him the most beautiful thing is "Love"."Love" ________ （make）the world go round. Without love there is no beauty.Still the artist continued his search. He asked a soldier the same question and his answer was "Peace". He said, "War is ugly and wherever you find peace, you will find beauty, faith and love." "How can I paint all of ________ （they）—Faith, Love and Peace? "He thought and thought. He was surprised that without ________ （think）where he was going, he had got to his familiar place. In the ________ （face）of his wife and children, he saw Love and Faith. "We are thinking of you all the time. We prayed（祈祷）you would return to us safely, "said his wife. The artist sat on his favourite old chair and his heart was at peace.The artist painted the most beautiful thing in the world and ________ （call）it "Home".3．语法填空The Earth is a planet. It and seven other planets go ________ the Sun. We ________ （call）the eight planets and the Sun the solar system. ________ first planet, next to the Sun, is Mercury （水星）. It is 58 million ________ （kilometer）from the Sun. Venus（金星）is the second planet from the Sun, and ________ （we）planet, the Earth, is the third. It is 150 million kilometers from the Sun.Jupiter（木星）, Saturn（土星）, Neptune（海王星）and Uranus（天王星）are all ________ （big）than the Earth, ________ Venus, Mars and Mercury are smaller than the Earth. Animals, trees and humans can only live ________ the Earth-the other planets in our solar system do not have air ________ water. Do any planets in other solar systems have ________ （life）? We don't know about it.4．用所给词的适当形式填空。

User ManualPLC-5 Programmable ControllerFlash Tool(Catalog Number 1785-L11B, -L20B, -L20C, -L20C15,-L20E, -L26B, -L30B, -L40B, -L40C, -L40C15, -L40E, -L40L,-L46B, -L46C15, -L60B, -L60C, -L60C15, -L60L, -L80B,-L80C, -L80C15, -L80E, -L86B)Use This Manual e this manual if you are responsible for performing a flashfirmware upgrade to a flash-based PLC-5® processor.This Manual Describes...This manual describes how to use the PLC-5 programmablecontroller flash tool to upgrade these products:•series E and later Enhanced PLC-5 processors•series E and later Ethernet® PLC-5 processors•series C and later ControlNet™ PLC-5 processorsThis manual also describes what to do if the flash upgrade does notcomplete successfully.The following publications contain additional information aboutAllen-Bradley PLC-5 processor products. To obtain copies, contactyour local Allen-Bradley office or distributor.For more information about:Read this publication:Publication number:Enhanced PLC-5 processors Enhanced and Ethernet PLC-5 ProgrammableControllers User Manual 1785-6.5.12Ethernet PLC-5 processorsControlNet 1.0 or 1.25 PLC-5 processors ControlNet PLC-5 Programmable ControllersUser Manual1785-6.5.14ControlNet 1.5 PLC-5 processors ControlNet PLC-5 Programmable ControllersPhase 1.5 User Manual1785-6.5.22For A-B Internal Use Only Publication 1785-6.2 March 19982Publication 1785-6.2 March 1998For A-B Internal Use OnlyBefore You Begin...Gathering What You Need Gather the following items before you begin using the flash upgrade.(Items marked with an asterisk (*) are included in the flash tool package you ordered.)•flash firmware upgrade tool disk*•PLC-5 processor whose firmware you want to update (with chassis and power supply)•personal computer with PLC-5 programming software installed •shielded serial cable (1784-CP10 or 1784-CP11)•this manual*•firmware revision label*•plug PROM(s) (if you require a plug PROM firmware update)*Running the Software You will copy the flash files to your hard ing the Flash ToolTo successfully use the flash tool, you need to complete seven steps:1.Prepare the PLC-5 processor.2.Install the flash tool.3.Perform the firmware upgrade.4.Apply the firmware revision label.5.Update the communication plug firmware, if necessary.6.Reconnect the PLC-5 processor.7.Uninstall the flash tool.!ATTENTION:You must install and run the PLC-5 flash tool on a computer booted up in DOS 6.22 or later. Do not use a DOS window running under Windows 95 or Windows NT .!ATTENTION:Pay strict attention to the flash tool procedure. Failure to follow this procedure exactly may result in an inoperable processor.3For A-B Internal Use Only Publication 1785-6.2 March 1998Step 1 - Preparing the PLC-5 Processor1.Save processor memory to your hard disk or to an EEPROM using yourPLC-5 programming software.2.Remove the processor battery cover and disconnect the battery. This resetsthe serial port to its default configuration so that the PLC-5 processor and theflash tool can communicate.3.Turn off processor power.4.Disconnect all cables (serial, DH+™, ControlNet, Ethernet, etc.) fromthe processor.5.Disconnect the coprocessor or PLC-5 Ethernet Interface module, if oneis connected.6.Turn the processor keyswitch to Program mode.7.Turn on processor power.8.Attach the shielded serial cable (1784-CP10 or 1784-CP11) betweenchannel 0 on the PLC-5 processor and the serial port on the computer.!ATTENTION:You must save the processor memoryto your hard disk or to an EEPROM. If you do not, thePLC-5 user memory will be lost because the flash procedure erases processor memory.4Publication 1785-6.2 March 1998For A-B Internal Use OnlyStep 2 - Installing the Flash Tool1.Boot the computer in DOS 6.22 or later. Do not use a DOS window running under Windows 95 or Windows NT . Verify that there is a DOS prompt on the screen.2.Create the following directory on the programming terminal’s hard drive by typing:md c:\flash3.Place the flash firmware upgrade tool diskette in the a: drive of the programming terminal. Copy the files from this diskette to the programming terminal hard drive by typing:4.On the programming terminal, change to the following directory containing the firmware files by typing:cd c:\flashStep 3 - Performing the Flash Upgrade Follow these steps to upgrade the processor firmware.1.Enter this command:The flash upgrade catalog number selection screen e the arrow keys to move the cursor to the catalog number of the flash upgrade you want to download to the processor and!ATTENTION:Make sure you install this application on the hard drive. Do not attempt to run it from the floppy disk; it will not work.!ATTENTION:Do not interrupt the flash download process by disturbing the processor or computer. Failure to do so may result in an inoperable processor.5For A-B Internal Use Only Publication 1785-6.2 March 19983.Select the appropriate script file by moving the cursor to the file name andLwwx_yz.ini4.On your computer, select the communication port (COM1 or COM2) by5.Make sure you have established a connection. This could take about 60seconds.Where:Represents:ww 11, 20, 26, 30, 40, 46, 60, 80, or 86depending on what flash upgrade you aretrying to download to the processor.x the type of flash upgrade to download:• B (for Enhanced)•L (for Extended-local I/O)• C (for ControlNet 1.0 or 1.25)• E (for Ethernet)•C15 (for ControlNet 1.5)y the series of the flash upgrade to download(see the disk label)z the revision of the flash upgrade to download(see the disk label)If you:The screen displays:established a connection information about the target processorThe processor’s PROC LED flashesred/green.did not establish a connection an error messageSee the following table.6Publication 1785-6.2 March 1998For A-B Internal Use OnlyIf you received an error:When the flash download begins:•the processor’s COMM LED blinks green •the processor’s PROC LED blinks red/green • a percent complete status (0 - 100) appears in the middle of the screen that indicates how much of the flash update is complete 6.Wait for the flash update to complete. The upgrade should take about 5 minutes to complete.When the download is complete the status bar will be at 100% and the screen will display this message: Processors Flash Update Completed. Press Any Key to Continue.If you received this error:Do this:Processor not connected. a.Make sure the keyswitch is in Program mode.b.Make sure the processor power is on.c.Check the serial cable.d.Verify the COM port being used by the computer.e.Return to item 3, step 3 on page 5 and repeat this section.Target processor does not match script file. a.Check the name of the script file.b.Make sure you are connected to the correct type and series of processor.c.Return to item 3, step 3 on page 5 and repeat this section.Firmware series stated in script file does not match PLC. a.Make sure you are connected to the correct type and series of processor.b.Return to item 3, step 3 on page 5 and repeat this section.No script files found. a.Make sure you copied all files from the firmware upgrade diskette to the c:\flash hard drive directory as described in item 2, step 2 on page 4.b.Make sure you selected the catalog number of the flash upgrade you want to download to the processor on the flash upgrade catalog number selection screen. Do not select the catalog number of the processor you currently have.c.Return to item 3, step 3 on page 5 and repeat this section.7For A-B Internal Use Only Publication 1785-6.2 March 19987.When the flash update is complete, cycle power to the processor.8.Disconnect the serial cable that you used to perform the flash upgrade.Step 4 - Applying the Firmware Revision LabelImportant: It is important to apply this label to your processor to ensure youreceive the correct version back if you send it in for repair.1.Turn off power to the processor.2.Remove the processor from the chassis.3.Apply the new firmware revision label in the space indicated by dotted lineson the product identification label. The following figure shows where toplace the label on a series E PLC-5 processors and on older PLC-5 processors.If the processor powers up with the:Do this:PROC LED solid red and CH1A DH+ LEDblinking green (normally)continue with the item 8, step 3 that follows this table.PROC LED flashing red/green(flash mode)Return to item 3, step 3 on page 5 and repeat this section.If you have repeated this item more than 3 times, send the processor toAllen-Bradley repair services.PROC LED solid red and CH1A DH+LED off8Publication 1785-6.2 March 1998For A-B Internal Use OnlyStep 5 - Updating the Communication Plug Firmware If you received communication plug PROM(s) with your order, you need to update your PROM(s) to complete the flash upgrade.1.Make sure you have already removed the battery cover and disconnected the battery and disconnected any cables attached to the processor.!ATTENTION:Printed circuit board components can be damaged during routine handling and installation. Follow these precautions to reduce static electricity discharges before you upgrade the processor firmware.•Handle the printed circuit board by the case or carrier without touching the pins or the edge connector.•Use a static-free workstation.•Connect the static-free workstation to ground through a minimum 200K ohm resistance.•Wear a grounded, conductive wrist strap with a minimum 200K ohm resistance.•Ground all tools before you begin to upgrade the firmware.•Control the relative humidity of the installation area. Ideal conditions are 40-60% relative humidity.9For A-B Internal Use Only Publication 1785-6.2 March 19982.Do one of the following:3.Remove the PROM, observing the orientation of the PROM notch. Replacethe plug PROM, checking to see that the PROM notch is correctly oriented.4.Reassemble and power up the processor.Step 6 - Reconnecting the PLC-5 Processor1.Reconnect the battery, any cables, and coprocessor or PLC-5 EthernetInterface module.2.Reload your program and make certain that it runs properly.(If you have any questions, call technical support at 440-646-6800.)If you have a:Do this:PLC-5/11, or -5/20 type processor a.Remove the four screws that hold the right sideplastic cover.b.Remove the cover.c.Remove the two large screws and washers located in the middle of the exposed circuit board.d.Separate the two processor boards by pulling theexposed circuit board at the backplane edge connectoraway from the metal cover as you would open a book.Notice the direction in which the battery cable is wrappedaround the nearby standoff.e.Disconnect the wires leading from the battery to thestake pins on the exposed circuit board.f.Disconnect the wires leading from the keyswitch to thestake pins on the exposed circuit board.See the figure at the left for the location of thecommunication plug PROM.Series D and earlier PLC-5/40C, -5/60C, or -5/80C processor Remove the phillips head screw near the channel 1B LED and gently remove the channel 1 communication plug. Beaware that the memory grounding clip may move or drop offfrom the cover mounting tab.41024。

矿物地质温度压力计（Geothermobarometry of minerals）二、矿物地质温压计的种类矿物地质温压计是以矿物特征为基础，根据矿物的不同性质，可将矿物地质温压计分为不同种类，常见者有:矿物稳定同位素地质温压计：从理论上讲，平衡矿物之间的稳定同位素分馏值是温度的函数；每一对平衡矿物的稳定同位素都能计算出来。

例如，石英–钠长石矿物对的同位素分馏温度计为：1000ln Qtz-Ab=0。

5106T-2。

矿物包裹体温压计:利用矿物中的流体、气体包裹体的均一温度、冰点等确定寄主矿物形成的温度以及校正压力.矿物离子交换温压计：利用矿物中或矿物之间离子交换性质建立起来的温压计。

目前地质研究中普遍使用该类温压计。

三、矿物离子交换温压计的理论基础简单地讲，离子交换地质温压计就是元素分配地质温压计，是利用元素分配远离建立起来的温压计。

自然界中的许多矿物，不论是地壳或地幔的矿物,还是陨石、月球或宇宙尘的矿物，其中绝大部分都是由两种或两种以上组分所构成的固溶体矿物。

共生的固溶体矿物，如果是处于平衡状态的话，又常常具有某一种或几种相同的元素(离子或原子）；另一方面，同一的元素也可以存在于同一矿物的不同结构位置中。

因此，共生矿物间或同一矿物的非等效结构之间、不同结构位置之间都可能存在离子或原子的交换问题，即元素的分配问题。

元素的分配问题受热力学定律 (Nernst定律）所支配。

假如把天然矿物看成理想溶体或近于理想溶体的话，那么某种元素在共生矿物之间或不同等效结构位置之间的分配数量之比，是受温度和压力的支配.因此,根据矿物的成分特点或矿物中元素的占位特点，反过来就可以推测矿物平衡时的温度和压力。

这就是矿物温压计的基本原理。

根据不同矿物共生组合，可写出矿物之间的多种化学计量关系,其中特别重要的有GASP(石榴子石–Al2SiO5–石英–斜长石)、GARB（石榴子石–黑云母）、GMPB(石榴子石–白云母–斜长石–黑云母)反应等。

Changing the Temperature Setpoint (cont’d…) Pressing and holding a key will cause the setpointtemperature to advance more quickly to a desired value. Three scanning speeds are provided: slow, medium and fast. The lower setpoint limit and upper setpoint limit are at -23 and 257F, respectively. While the min. and max. setting are changeable, it is not advised as it may result in damage to the calibrator.Heat-Up/Cool-Down Transition Time Testing/Calibrating Temperature ProbesWhen calibrating probes at different temperature points, start at the lowest temperature and work up to thehighest temperature. Do not jump up and down from a very hot temperature to a relatively cooler temperature. This will reduce the time it takes for the probe well to re-stabilize after you change the setpoint. When placing probes into the well, make sure the probe tip goes all the way down to the bottom of the probe well, the full 4.5". This will insure the degree of highest accuracy possible when taking your reading.After calibrating each probe, remove it from the well and place it on a protected surface to cool. If you have another probe to calibrate, place it into the probe well and allow the calibrator a few minutes to re-stabilize.WGS/START HERE ARRO 5WGS/START HERE ARRO6CL1500Bench-Top Dry Block Calibrator***********************®It is the policy of OMEGA Engineering, Inc. to comply with all worldwide safety and EMC/EMI regulations that apply. OMEGA is constantly pursuing certification of its products to the European New Approach Directives. OMEGA will add the CE mark to every appropriate device upon certification.The information contained in this document is believed to be correct, but OMEGA accepts no liability for any errors it contains, and reserves the right to alter specifications without notice.WARNING: These products are not designed for use in, and should not be used for, human applications.MQS4695/0417Overheat Reset SwitchIf the unit is operated at high temperatures in elevated ambient temperatures, an overheat condition may occur. In an overheat situation a mechanical reset switch inside the unit will pop and open the heater circuit. The controller will still have power. While the controller will be demanding heat from the heater, the process temperature will fall or rise continuously until it equalizes with room temperature. If an overheatcondition occurs, let the unit cool off for one hour. If this does not correct the problem, contact the factory.turn the calibrator off until completing the cool-down procedure.When you have completed working with the calibrator you must cool the unit down to ambient temperature if you intend to move your unit and/or return to storage.Serial Cable ConnectionThe CL1500 features a serial port that allowsbi-directional data transfer via a three conductor cable consisting of signal ground, receive input, and transmit output. It is recommended that less than fifty feet of cable be used between the computer and this instrument. For detailed information on RS232 Communication and Software refer to Section 4 in the User’s GuideConnecting the CL1500 to a Computer’s Serial Port/manuals/manualpdf/M4695.pdfTime -5°C (23°F) to 23°C (73.4°F) 1 minute 23°C (73.4°F) to 100°C (212°F) 3 minutes 100°C (212°F) to 125°C (257°F)5 minutesHeating TimesTime 125°C (257°F) to 100°C (212°F) 1 minute 100°C (212°F) to 0°C (32°F) 8 minutes 0°C (32°F) to -5°C (23°F)4 minutesCooling TimesWARRANTY/DISCLAIMEROMEGA ENGINEErING, INC. warrants this unit to be free of defects in materials and workmanship for a period of 13 months from date of purchase. OMEGA’s WArrANTY adds an additional one (1) month grace period to the normal one (1) year product warranty to cover handling and shipping time. T his ensures that OMEGA’s customers receive maximum coverage on each product.If the unit malfunctions, it must be returned to the factory for evaluation. OMEGA’s Customer Service Department will issue an Authorized return (Ar) number immediately upon phone or written request. Upon examination by OMEGA, if the unit is found to be defective, it will be repaired or replaced at no charge. OMEGA’sWArrANT Y does not apply to defects resulting from any action ofthe purchaser, including but not limited to mishandling, improper interfacing, operation outside of design limits, improper repair, or unauthorized modification. T his WArrANT Y is VOID if the unit shows evidence of having been tampered with or shows evidence of having been damaged as a result of excessive corrosion; or current, heat, moisture or vibration; improper specification; misapplication; misuse or other operating conditions outside of OMEGA’s control. Components in which wear is not warranted, include but are not limited to contact points, fuses, and triacs.OMEGA is pleased to offer suggestions on the use of its various products. However, OMEGA neither assumes responsibility for any omissions or errors nor assumes liability for any damages that result from the use if its products in accordance with information provided by OMEGA, either verbal or written. OMEGA warrants only that the parts manufactured by the company will be as specified and free of defects. OMEGA MAKES NO OTHER WARRANTIES OR REPRESENTATIONS OF ANY KIND WHATSOEVER, EXPRESSED OR IMPLIED, EXCEPT THAT OF TITLE, AND ALL IMPLIED WARRANTIES INCLUDING ANY W ARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE HEREBY DISCLAIMED. LIMITATION OF LIABILITY: The remedies of purchaser set forth herein are exclusive, and the total liability of OMEGA with respect to this order, whether based on contract, warranty, negligence, indemnification, strict liability or otherwise, shall not exceed the purchase price of the component upon which liability is based. In no event shall OMEGA be liable for consequential, incidental or special damages.CONDITIONS: Equipment sold by OMEGA is not intended to be used, nor shall it be used: (1) as a “Basic Component” under 10 CFr 21 (NrC), used in or with any nuclear installation or activity; or (2) in medical applications or used on humans. Should any Product(s) be used in or with any nuclear installation or activity, medical application, used on humans, or misused in any way, OMEGA assumes no responsibility as set forth in our basic WArrANT Y/DISCLAIMEr language, and, additionally, purchaser will indemnify OMEGA and hold OMEGA harmless from any liability or damage whatsoever arising out of the use of the Product(s) in such a manner.RETURN REQUESTS/INQUIRIESDirect all warranty and repair requests/inquiries to the OMEGA Customer Service Department. BEFOrE rE UrNING ANY PrODUC (S) O OMEGA, PUrCHASEr MUS OB AIN AN AUTHOrIZED rETUrN (Ar) NUMBEr FrOM OMEGA’S CUSTOMEr SErVICE DEPArT MENT (IN OrDEr T O AVOID PrOCESSING DELAYS). T he assigned Ar number should then be marked on the outside of the return package and on any correspondence.FOr WARRANTY rETUrNS, please have the followinginformation available BEFOrE contacting OMEGA:1. Purchase Order number under which the product was PUrCHASED,2. Model and serial number of the product under warranty, and3. repair instructions and/or specific problems relative to the product.FOr NON-WARRANTY rEPAIrS, consult OMEGA for current repair charges. Have the following information available BEFOrE contacting OMEGA:1. Purchase Order number to coverthe COST of the repair or calibration,2. Model and serial number of the product, and3.r epair instructions and/or specific problems relative to the product.OMEGA’s policy is to make running changes, not model changes, whenever an improvement is possible. This affords our customers the latest in technology and engineering.OMEGA is a registered trademark of OMEGA ENGINEErING, INC.© Copyright 2017 OMEGA ENGINEErING, INC. All rights reserved. T his document may not be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form, in whole or in part, without the prior written consent of OMEGA ENGINEErING, INC.Available ModelsModel No.*Hole Size CL1500 Standard See Fig. 2CL1500M Metric * Add suffix -230 for 230 Vac Models.Probe Wells (With Dimensions)MountingMount the unit on a bench, table top or shelf in ahorizontal position and operate at least ten inches from any air obstructions to the fan, front panel, rear panel, bottom and top of the unit, in an ambient environment between the specified 0 to 45°C (32 to 113°F).Ambient TemperatureThe calibration block of the CL1500 can achieve any temperature within the specified temperature range of -5 to 125°C (+23 to 257°F) when being operated in normal ambient temperature 23°C (72°F) environments. As long as the ambient temperature does not exceed 25°C (75°F), the block will achieve its lower limit temperature of -5°C (23°F). The minimum block temperature the unit can achieve is proportionallyhigher with increased ambient temperature. An increase in Ambient temperature of 1°C (1.8°F) above the 23°C (72°F) increases the minimum probe well temperature by approximately 0.8°C (1.4°F).WGS/START HERE ARROWGS/START HERE ARRO 2WGS/START HERE ARRO34Using This Quick Start ManualUse this Quick Start Manual with your CL1500 Series Bench-Top Dry Block Calibrator for easy installation and basic operation. For detailed information, refer to the User’s Guide (Manual Number M4695).PRECAUTIONS:• F ollow all safety precautions and operating instructions outlined in this quick start and accompanying User’s Guide.• N ever leave your calibrator unattended when in use.• K eep out of reach of all children.• N ever touch the probe well or probes when hot without proper protection.• N ever place any objects other than temperature probes in the well.• D o not operate in flammable or explosive environments.• N ever operate with a power cord other than the one provided with your unit.• T urn unit off and disconnect main power cord before attempting any maintenance or fuse replacement.• N ever disconnect main power cord or main power source when unit is still hot.• D o not connect and or operate this unit to a non-grounded, non-polarized outlet or power source.• T his unit is intended for indoor use only. Avoid exposure to moisture or high humidity.• Never operate the unit outside.• D o not return your unit to storage when hot, allow unit to cool down to ambient temperature.General InformationThe Model CL1500 is a portable, rugged, bench-top, hot/cold dry block calibration source with a built-in precision PID digital controller. Thecalibrator is used to test and calibrate temperature probes of various diameters. The calibration block has 6 holes to accommodate probes of varying diameter. It is available in both standard and metric versions. It can be set to any temperature between -5 to 125°C (+23 to 257°F).The Effect of Increased Ambient Temperature onOperating TemperaturePower ConnectionInternational (230 Vac~, 50/60 Hz Models only)On “-230Vac” models an International style power cord with the proper color code and approvals is provided with stripped wire ends for connection to the proper connector used in your country or local area, this connector is not provided. Make sure when installing your connector to the wire ends that the ground connection has been made.Certification: CE (CL1500 ~230VAC only)and inside the calibrator’s enclosure when connected to the AC mains supply. Do not remove the top or bottom cover of the calibrator for any reason.Front Panel Controls and IndicatorsFront PanelProcess TemperatureThis field displays the current temperature of the calibration block.Setpoint TemperatureThis field displays the desired calibration block temperature. Once the block reaches this desired temperature, both displays will read the same value.Parameter/Access Key:Used to index through parameters or to access Menu levels.Raise Key: Used to scroll up through available parameter settings, increase values or change menu levels (Hold for fast-step progression).L ower Key: Used to scroll down through available parameter settings, decrease values or change menu levels (Hold for fast-step progression).M ode Key: This key is inactive.Press to save settings and exit a menu level Back PanelChanging the Temperature Setpoint The CL1500’s upper display indicates the calibration block temperature known as (PV)Process Variable, while the lower display indicates the programmed setpoint known as (SV) Setpoint Variable. Changes to the setpoint, units of measure and communication settings are made via the raise and lower keys.RAISE KEYP ARAMETER/ACCESS KEYCALIBRA TIONFANVENTAC POWER INPUT。

CÁMARAS JABRA / HD 1080P MANUAL RÁPIDO INICIO EN 10 PASOSIMPORTANTE:Para el uso de este manual y de Google Meet es necesario de uso del navegador Google Chrome, instalado en los equipos informáticos.Cámaras Jabra instaladas en el techo del aula:Para un correcto funcionamiento de las cámaras, no se deberán manipular estas ni físicamente ni por software. Se han situado en posiciones y valores óptimo para su uso en la docencia.No se ha de abrir en ningún momento ningún otro programa de cámaras ya que se pueden desconfigurar los parámetros de las cámaras Jabra Panacast instaladas.Cámaras HD 1080p instaladas en la mesa:Están instaladas en las posiciones óptimas para su uso, no obstante, y en alguna ocasión justificada, se podrán manipular para que la imagen una vez iniciada la sesión de Google Meet se adapte a las marcas negras de la pizarra.Pasos a realizar:1º) Datos a comprobar antes de abrir sesión de Google Meet:- El Pc está encendido- Ratón y teclado funcionan correctamente- En la Cámara Jabra del techo, aparecen 2 luces azules encendidas.- Si es la Cámara HD 1080p de la mesa puede encenderse una luz, normalmente verde- Sonido del ordenador: cuidado si los altavoces están a volumen muy alto. Asegúrese que elaudio del ordenador está a un volumen alto regular el sonidosegún el aula (icono parte inferior derecha del escritorio, junto a la fecha) - Asegurarse que la pantalla está duplicada y se ve en la pantalla del cañón lo mismo que en la pantalla.- Asegurarse que la pantalla está Duplicada:o Edificio Ada Byron y Torres Quevedo seguir estos pasos:Pulsar la “tecla de Windows” y la letra “P” aparecerá un menú en la partesuperior izquierda de la pantalla:y seleccionar “Duplicado”.o Edificio Betancourt no hace falta hacer este paso.2º) Abrir el navegador Google Chrome (Modo “incógnito”) para que no se guarde la configuración anterior al cerrar esta aplicación.Pulsaremos 2 veces en el botón izquierdo del ratón, sobre el icono o bien,pulsaremos 1 vez en el botón izquierdo del ratón, donde también aparece este icono(en Windows 7 no está en la barra de tareas)3º) Iniciamos sesión con nuestro usuario de @unizar.es(podremos grabar la reunión en el Google Meet de la clase)Podemos iniciar la sesión de una de las dos formas:a) Pulsamos sobre el icono “A plicaciones de Google” y después pulsamos sobre GoogleMeet para iniciar la sesión.b) Podemos ir directamente a nuestra sesión a través de Calendar4º) Una vez que estemos en Google Meet pulsar sobre5º) Podemos poner el código o alias de la reunión y/o pulsar sobre “Continuar”6º) Pulsar sobre el cuadro superior: “ quiere usar el micro”El mensaje inferior se cerrará automáticamente después de pulsar “Permitir”7º) Antes de unirse a la reunión se debe configurar la resolución: en el Meet, en los 3 puntitos“Más Opciones” seleccionar la última:y desde allíEn la parte izquierda, en los apartados:Debe cambiarse “Automático” ·por “Alta definición (720p)”:Pulsar sobre la palabra “Automático” y seleccionar en el menú la opción “Alta definición (720)”En el apartado “Resolución de Recepción, seguir el mismo procedimiento8º) En la ventana “Configuración” se debe comprobar que el micro está activo y funciona.Para ello ir a “Audio” y comprobar que al hablar aparecen unos puntitos en la parte derecha del audio.También podemos probar el sonido si pulsamos sobre la palabra “Probar” de este mismo apartado de “Audio”.9º). Después cerrarem,os esta ventana por medio de la .10º). Ya es posible iniciar el streaming de la clase.OBSERVACIONES:Para comunicar cualquier incidencia con las cámaras Jabra instaladas manden un correo electrónico:***********************Fuentes: En este manual se ha utilizado fuentes e imágenes de la web www.jabra.esManual realizado por Javier Aurelio Tomás (Técnico E. de Medios Informáticos y Audiovisuales Vers.8)。

Clontech Laboratories, Inc.SMARTer® RACE 5’/3’Kit User ManualCat. No(s). 634858, 634859(022714)Clontech Laboratories, Inc.A Takara Bio Company1290 Terra Bella Avenue, Mountain View, CA 94043, USAU.S. Technical Support: tech@United Asia Pacific Europe Japan Page 1 of 30I. Introduction (4)II. List of Components (7)III. Additional Materials Required (8)IV. Primer Design (9)A. Primer Sequence (9)B. Additional Considerations for Design (10)C. Location of Primer Sequences within Genes (10)D. Nested Primers (10)V. Generating RACE-Ready cDNA (11)A. General Considerations (11)B. Preparation and Handling of Total and Poly A+ RNA (11)C. Assessing RNA Template Quality (12)D. Protocol: First-Strand cDNA Synthesis (13)VI. Rapid Amplification of cDNA Ends (RACE) (15)A. Things You Should Know Before Starting RACE PCR Reactions (15)B. Protocol: Rapid Amplification of cDNA Ends (RACE) (15)VII. Characterization of RACE Products (17)A. Protocol: Gel Extraction with the NuceloSpin Gel and PCR Clean-Up Kit (17)B. Protocol: In-Fusion Cloning of RACE Products (18)C. Sequencing RACE Products (19)VIII. References (20)Appendix A. Troubleshooting Guide (21)A. Troubleshooting Touchdown PCR (21)B. Multiple Band RACE Products (23)C. Other Specific Problems (25)Ap pendix B. Detailed Flow Chart of 5’ RACE (27)Appendix C. Detailed Flow Chart of 3’ RACE (28)Appendix D. 5’-RACE cDNA Amplification with Random Primers (29)A. Protocol: First-Strand cDNA Synthesis with Random Priming (29)Figure 1. Mechanism of SMARTer cDNA synthesis (4)Figure 2. Overview of the SMARTer RACE procedure.. (6)Figure 3. The relationship of gene-specific primers to the cDNA template. (9)Figure 4. 5'- and 3'-RACE sample results (22)Figure 5. Detailed mechanism of the 5'-RACE reactions. (27)Figure 6. Detailed mechanism of the 3'-RACE reactions. (28)Table of TablesTable 1. Additional 5'-RACE Sequence Obtained with SMART Technology (5)Table 2. Setting up 5'- and 3'-RACE PCR Reactions (15)Table 3. Troubleshooting Guide: Other Specific Problems (25)I. IntroductionThe SMARTer RACE 5’/3’ Kit provides a method for performing both 5’- and 3’-rapid amplification of cDNA ends (RACE). The SMARTer RACE 5’/3’ Kit includes our SMARTer II A Oligonucleotide and SMARTScribe™Reverse Transcriptase, which provides better sensitivity, less background and higher specificity than previouskits. This powerful system allows you to amplify the complete 5’ sequence of your target transcript from as little as 10 ng of total RNA. The cornerstone of SMARTer RACE cDNA synthesis is SMART™ technology, which eliminates the need for problematic adaptor ligation and lets you use first-strand cDNA directly in RACE PCR, a benefit that makes RACE far less complex and much faster (Chenchik et al., 1998). Additionally, the SMARTer RACE Kit exploits Clontech’s technology for suppression PCR & step-out PCR to increase the sensitivity andreduce the background of the RACE reactions. You can use either poly A+ or total RNA as starting material for constructing full-length cDNAs, even of very rare transcripts.The SMARTer RACE 5’/3’ Kit is an improved version of our original SMARTer RACE cDNA Amplification Kit, designed to accommodate larger RNA input volumes and perform more efficiently on challenging targets(e.g., those that are long, GC-rich, etc.). RACE PCR products are amplified with our highly robust SeqAmp™DNA Polymerase, and cloned into the linearized pRACE vector with In-Fusion® HD Cloning. The In-Fusion HD Cloning Kit, NucleoSpin Gel and PCR Clean-Up Kit, and Stellar™ Competent Cel ls are included for yourconvenience in cloning RACE products.SMART technology provides a mechanism for generating full-length cDNAs in reverse transcription reactions (Zhu et al., 2001). This is made possible by the joint action of the SMARTer II A Oligonucleotide andSMARTScribe Reverse Transcriptase. When the SMARTScribe RT reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA (Figure 1).Figure 1. Mechanism of SMARTer cDNA synthesis.First-strand cDNA synthesis is primed using a modified oligo (dT) primer. After SMARTScribe Reverse Transcriptase (RT) reaches the end of the mRNA template, it adds several nontemplated residues. The SMARTer IIA Oligonucleotide anneals to the tail of the cDNA and serves as an extended template for SMARTScribe RT.The SMARTer II A Oligonucleotide contains a terminal stretch of modified bases that anneal to the extended cDNA tail, allowing the oligo to serve as a template for the RT. SMARTScribe RT switches templates from the mRNA molecule to the SMARTer oligo, generating a complete cDNA copy of the original RNA with the additional SMARTer sequence at the end. Since the template switching activity of the RT occurs only when the enzyme reaches the end of the RNA template, the SMARTer sequence is typically only incorporated into full-length, first-strand cDNAs. This process guarantees that the use of high quality RNA will result in the formation of a set of cDNAs that have a maximum amount of 5’ sequence (Table I).Table 1. Additional 5'-RACE Sequence Obtained with SMART TechnologyHuman gene Size of mRNA(kb)Additional sequence(bp)*Matches genomicsequencesPiccolo presynaptic cytomatrix protein 20.29 +59 yesDynein, cytoplasmic 1, heavy chain 1 14.36 +36 yesPolycystic kidney disease 1 14.14 +21 yesSolute carrier family 1 12.02 +73 yes Microtubule-associated protein 1A 10.54 +13 yesSpectrin, beta, non-erythrocytic 10.24 +32 yesTransferrin receptor 5.0 +25 yesInterferon-α receptor 2.75 +17 yesSmooth muscle g-actin 1.28 +31 yesFollowing reverse transcription, SMART technology allows first-strand cDNA to be used directly in 5’- and3’-RACE PCR reactions. Incorporation of universal primer binding sites in a single-step during first-strand cDNA synthesis eliminates the need for tedious second-strand synthesis and adaptor ligation. This simple and highly efficient SMARTer cDNA synthesis method ensures higher specificity in amplifying your target cDNA. Suppression PCR & step-out PCR techniques are used in combination with SMARTer technology to decrease background amplification in RACE PCR.Requirements for SMARTer RACE cDNA AmplificationThe only requirement for SMARTer RACE cDNA amplification is that you know at least 23–28 nucleotides (nt) of sequence information in order to design gene-specific primers (GSPs) for the 5’- and 3’-RACE reactions. (Additional sequence information will facilitate analysis of your RACE products.) This limited requirement makes SMARTer RACE ideal for characterizing genes identified through diverse methods, including cDNA subtraction, differential display, RNA fingerprinting, ESTs, library screening, and more.Uses of SMARTer RACE cDNA AmplificationSMARTer RACE cDNA amplification is a flexible tool—many researchers use this kit in place of conventional kits to amplify just the 5’ or 3’ end of a particular cDNA. Others perform both 5’- and 3’-RACE, and many then go on to clone full-length cDNAs using one of the two methods described in the latter part of this protocol. In many cases, researchers obtain full-length cDNAs without ever constructing or screening a cDNA library.Figure 2. Overview of the SMARTer RACE procedure.Detailed flow charts of the SMARTer RACE mechanisms can be found in Appendices B & C. Alternatively, you can obtain the sequences of the extreme ends of the transcript by sequencing the 5’ end of the 5’ product and the 3’ end of the 3’ product. Using this information, you can design 5’ and 3’ gene-specific primers to use in LD PCR with the 5’-RACE-Ready cDNA as template to generate the full-length cDNA.Note that with the cloned RACE fragments you can use a restriction site in an overlapping region to construct a full-length cDNA by subcloning, or design new GSPs to generate PCR products compatible with In-Fusion cloning.II. List of ComponentsThis section lists the components for Cat. No. 634858, a 10 reaction kit. The larger, 20 reaction kit (Cat. No.634859) contains two of every item listed below.SMARTer RACE 5’/3’ Kit Components (Cat. No. 634860) (Not sold separately)Store SMARTer II A Oligonucleotide and Control Mouse Heart Total RNA at –70°C. Store all other components at –20°C.∙First-Strand cDNA Synthesiso10 µl SMARTer II A Oligonucleotide (24 μM)o10 µl 3' RACE CDS Primer A (12 μM)o10 µl 5' RACE CDS Primer A (12 μM)o10 µl 10X Random Primer Mix (20 μM)o40 µl 5X First-Strand Buffer (RNAse-Free)o 5 µl Dithiothreitol (DTT) (100 mM)o 1 ml Deionized H2Oo10 µl RNase Inhibitor (40 U/µl)o20 µl SMARTScribe Reverse Transcriptase (100 U/µl)o10 µl dNTP mix (20 mM)∙5’- and 3’-RACE PCRo400 µl 10X Universal Primer A Mix (UPM)o50 µl Universal Primer Short (10 µM)o 5 µl Control Mouse Heart Total RNA (1 µg/µl)o25 µl Control 5'-RACE TFR Primer (10 µM; designed for compatibility with In-Fusion cloning)o25 µl Control 3'-RACE TFR Primer (10 µM; designed for compatibility with In-Fusion cloning) ∙In-Fusion Cloningo10 µl Linearized pRACE (50 ng/µl)∙General Reagentso 2 tubes Tricine-EDTA Buffer (1 ml each)SeqAmp DNA Polymerase (Cat. No. 638504)Store all components at –20°C.∙50 μl SeqAmp DNA Polymerase∙ 1.25 ml SeqAmp PCR Buffer (2X)In-Fusion HD Cloning Kit (Cat. No. 639648) (Not sold separately)Store all components at –20°C.∙20 μl 5X In-Fusion HD Enzyme Premix∙ 5 μl pUC19 Control Vector, linearized (50 ng/μl)∙10 μl 2 kb Control Insert (40 ng/μl)NucleoSpin Gel and PCR Clean-Up Kit (Cat. No. 740609.10) (Not sold separately)Store all components at room temperature∙10 ml Binding Buffer NTI∙ 6 ml Wash Buffer NT3 (concentrate)∙ 5 ml Elution Buffer NE (5 mM Tris/HCl, pH 8.5)∙10 NucleoSpin Gel and PCR Clean-Up Columns (yellow rings)∙10 Collection Tubes (2 ml)Stellar Competent Cells (Cat. No. 636763)Store Stellar Competent Cells at –70°C. Store all other components at –20°C.∙10 tubes Stellar Competent Cells (100 µl/tube)∙10 tubes SOC Medium (1 ml/tube)∙10 µl pUC19 Vector (0.1 ng/µl)III. Additional Materials RequiredIf your RNA template is from a non-eukaryotic organism and lacks a polyadenylated tail, you can add one prior to first-strand 3’-cDNA synthesis using the following enzyme:∙Poly(A) Polymerase (Takara Bio Cat. No. 2180A)The following materials are required for In-Fusion cloning and transformation, but not supplied: ∙Ampicillin (100 mg/ml stock) or other antibiotic required for plating the In-Fusion reaction∙LB (Luria-Bertani) medium (pH 7.0)∙LB/antibiotic plates∙SOC mediumThe following material is required for the NucleoSpin Gel and PCR Clean-Up Kit, but not supplied: ∙96–100% ethanolIV. Primer DesignA. Primer SequenceGene-Specific Primers (GSPs) should:∙be 23–28 nt to ensure specific annealing∙be 50–70% GC∙have a T m≥65°C; best results are obtained if T m >70°C, which enables the use of touchdown PCR.(T m should be calculated based upon the 3’ (gene-specific) end of the primer, NOT the entire primer.) ∙not be complementary to the 3’-end of the Universal Primer MixLong primer = 5’–TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT–3'Short primer = 5’–CTAATACGACTCACTATAGGGC–3’∙be specific to your gene of interest∙both have 15 bp overlaps with the vector at their 5’ end s (i.e., add the sequenceGATTACGCCAAGCTT to the 5’ end s of both GSPs’ sequences; see details below) The relationship of the primers used in the SMARTer RACE reactions to the template and resultingRACE products are shown in detail in Figure 3.For the complete SMARTer RACE protocol, you will need at least two GSPs: an antisense primer for the5’-RACE PCR and a sense primer for the 3’-RACE PCR. If you are performi ng only 5’- or 3’-RACE,you will only need one GSP. In our experience, longer GSPs with annealing temperatures above 70°Cgive more robust amplification in RACE, particularly from difficult samples; however, there is generallyno advantage to using primers with gene-specific sequence longer than 30 nt.Successful In-Fusion cloning requires a 15 bp overlap with the linearized vector. Given this, you will needto add the sequence GATTACGCCAAGCTT to the 5’-end of your 5’ and 3’ GSPs to facilitate In-Fusioncloning of your RACE PCR products. This specific sequence is in addition to the 22 nt gene-specificsequence described above. The provided linearized pRACE vector already contains this overlap with theUniversal Primer A Mix included for PCR, and adding this sequence to the 5’-end of your GSPs willcomplete the necessary overlap for the cloning reaction. Please note that the In-Fusion User Manualcontains only general primer recommendations that should not be used for this particular protocol.Figure 3. The relationship of gene-specific primers to the cDNA template.This diagram shows a generalized first-strandcDNA template. This RNA/DNA hybrid does not precisely represent either the 5’- or 3’-RACE-Ready cDNAs. For a detailedlook at those structures, see Appendices B & C. Note that the gene-specific primers designed here contain tails with In-Fusionhomology, and also produce overlapping RACE products. This overlap permits the use of the primers together in a control PCRreaction. Additionally, if a suitable restriction site is located within this region, it will be possible to construct the full-lengthcDNA by subcloning.B. Additional Considerations for DesignThe primers shown in Figure 3 will create overlapping 5’- and 3’-RACE products. If a suitable restriction site is located in the region of overlap, the fragments can subsequently be joined by restriction digestion and ligation to create the full-length cDNA. If no suitable restriction sites are available, you canalternately design new GSPs suitable for multi-fragment In-Fusion cloning. By designing primers thatgive a 100–200-bp overlap in the RACE products, you will also be able to use the primers together as an internal positive control for the PCR reactions. However, it is not absolutely necessary to use primers that give overlapping fragments. In the case of large and/or rare cDNAs, it may be better to use primers that are closer to the ends of the cDNA and therefore do not create overlapping fragments. The primersthemselves can overlap (i.e., be complementary).C. Location of Primer Sequences within GenesWe have had good success using the SMARTer RACE Kit to amplify 5’ and 3’ cDNA fragments thatextend up to 6.5 kb from the GSP binding sites. Nevertheless, for optimum results, we recommendchoosing your primers so that the 5’- and 3’-RACE products will range from 1–3 kb in length. If you are working with an annotated genome, we suggest using NCBI’s Primer-BLAST to aid in your design for each transcript.D. Nested PrimersWe recommend that you do not use nested PCR in your initial experiments. The UPM Primer and a GSP will usually generate a good RACE product with a low level of nonspecific background. However, nested PCR may be necessary in some cases where the level of background or nonspecific amplification in the 5’- or 3’-RACE reaction is too high with a single GSP. In nested PCR, a primary amplification isperformed with the outer primers and, if a smear is produced, an aliquot of the primary PCR product is re-amplified using the inner primers. The SMARTer RACE protocols include optional steps indicatingwhere nested primers can be used. The Universal Primer Short (provided with the kit) can be used forbo th 5’- and 3’-RACE with nested primers.Nested gene specific primers (NGSP) should be designed according to the same guidelines discussedabove. If possible, nested primers should not overlap with the outer gene-specific primers; if they mustoverlap due to limited sequence information, the 3’ end of the inner primer should have as much unique sequence as possible. Additionally, your nested primers should also contain the 15 bp overlap required for In-Fusion cloning.V. Generating RACE-Ready cDNAPLEASE READ THE ENTIRE PROTOCOL BEFORE STARTINGA. General Considerations∙We recommend using the Tricine-EDTA Buffer provided in the kit to resuspend and dilute your cDNA samples throughout the protocols in this user manual, because Tricine buffers maintain theirpH at high temperature better than Tris-based buffers. Tris-based buffers can lead to low pHconditions that degrade DNA.∙Resuspend pellets and mix reactions by gently pipetting the solution up and down or by flicking the bottom of the tube. Always spin tubes briefly prior to opening to collect the contents at the bottom ofthe tubes.∙Perform all reactions on ice unless otherwise indicated.∙Add enzymes to reaction mixtures last.∙Ethidium bromide (EtBr) is a carcinogen. Use appropriate precautions when handling and disposing of this reagent. For more information, see Molecular Cloning: A Laboratory Manual by Sambrook &Russell (2001).B. Preparation and Handling of Total and Poly A+ RNA1. General PrecautionsThe integrity and purity of your total or poly A+ RNA starting material is an important element inhigh-quality cDNA synthesis. The following precautions will help you avoid contamination anddegradation of your RNA:∙Have a separate bench and/or pipette set dedicated to RNA work, free of RNasecontamination.∙Wear gloves throughout to protect your RNA samples from nucleases.∙Use freshly deionized (e.g., MilliQ-grade) H2O directly, without treatment with DEPC(diethyl pyrocarbonate). Takara Bio also offers RNase-Free Water (Cat. No. 9012).∙Use only single-use plastic pipettes and pipette tips. Filter tips are recommended.2. RNA IsolationClontech® offers several kits for isolating total or poly A+ RNA from a variety of sources:Purified Product Starting Material Product Cat. #Total RNA Plant or fungal samples NucleoSpin RNA Plant 740949.50mRNA Total RNA derived fromcultured cells or animal tissuesMagnosphere UltraPuremRNA Purification Kit9186Many procedures are available for the isolation of poly A+ RNA (Farrell, 1993; Sambrook et al., 2001).3. RNA PurityThe purity of RNA is the key factor for successful cDNA synthesis and SMARTer RACE. Thepresence of residual organics, metal ions, salt or nucleases in your RNA sample can have a largeimpact on downstream enzymatic applications by inhibiting enzymatic activity or degrading theRNA. We strongly recommend checking the stability of your RNA to ensure that it is free ofcontaminants. Impurities such as salt or organic contaminants can be removed by repeatedethanol precipitation, subsequent washing with 80% ethanol and the complete removal of allremaining ethanol.Since RNA stability is a good indicator of RNA purity, we strongly recommend checking thestability of your RNA to ensure that it is free of contaminants.Incubate a small portion of your RNA at 37°C for 2 hours, then compare the sample to a duplicatecontrol stored at –70°C. If the sample incubated at 37°C shows a lower 28S:18S ratio than thecontrol or a significant downward shift on a formaldehyde agarose gel, the RNA may havenuclease contaminants (see Section V.C., below, for methods for assessing RNA quality).If your RNA template is from a plant or some other species with high pigment levels, pleasepay special attention to polysaccharide/pigment contamination. Polysaccharides/pigments arehard to remove and can’t be detected on the agarose gel. These glycoproteins might interfere withprimer binding sites of RNA during the first-strand cDNA synthesis leading to reduced cDNAyield.C. Assessing RNA Template Quality1. Methods for Assessing Total RNA Integrity∙Detection with the Agilent 2100 BioAnalyzer (Agilent Technologies, CA):This microfluidics-based technology, which provides an alternative to traditional gel-based analysis, requires only 2–7 ng of RNA per analysis. We recommend using RNAsamples with an RNA Integrity Number (RIN) of 7 or higher. In addition to assessingRNA quality, this automated system provides a good estimate of RNA concentration.∙If you do not have access to an Agilent 2100 BioAnalyzer, you can visualize your RNA on a denaturing formaldehyde agarose gel under UV light. The theoretical 28S:18S ratiofor eukaryotic RNA is approximately 2:1. If the 28S:18S ratio of your RNA is less than 1,your RNA template is not suitable for SMARTer RACE. When visualizing RNA usingEtBr, you need at least 0.5–1 µg of total RNA. Alternatively, SYBR® Green II or SYBRGold dyes (Molecular Probes; Eugene OR), allow you to detect as little as 1 or 2 ng ofRNA on your gel, respectively.2. Methods for Assessing mRNA IntegrityAll of the methods mentioned above can be used to assess the quality of your mRNA. However,because mRNA does not contain strong ribosomal bands, the assessment of its quality will besomewhat subjective. Typically, mRNA appears as a smear between 0.5 kb to 6 kb, with an areaof higher intensity around 1.5 and 2 kb. This size distribution may be tissue or species-specific. Ifthe average size of your mRNA is less than 1.5 kb, it could be an indication of degradation.D. Protocol: First-Strand cDNA SynthesisThe two 20 µl reactions described in the protocol convert 10 ng–1 µg of total or poly A+ RNA intoRACE-Ready first-strand cDNAWe recommend that you use poly A+ RNA whenever possible. However, if you have less than 50 µg of total RNA we do not recommend purification of poly A+ RNA because the final yield will be too small to effectively analyze the RNA quantity and quality.We strongly recommend that you perform a positive control cDNA synthesis using the includedMouse Heart Total RNA in addition to your experimental reactions.NOTE: If your RNA template is from a non-eukaryotic organism and/or lacks a polyadenylated tail,follow the protocol for 5’-first-strand cDNA synthesis with random primers in Appendix D.For 3’-first-strand cDNA synthesis, add a poly(A) tail using Poly(A) Polymerase (Takara Cat. No.2180A), and proceed with the following protocol.IMPORTANT:∙Prior to cDNA synthesis, please make sure that your RNA is intact and free of contaminants (see Section V.C. Assessing the Quality of the RNA Template).∙Do not change the volume of any of the reactions. All components have been optimized for the volumes specified.1.Prepare enough of the following Buffer Mix for all of the 5’- and 3’-RACE-Ready cDNA synthesisreactions plus 1 extra reaction to ensure sufficient volume. Mix the following reagents and spinbriefly in a microcentrifuge, then set aside at room temperature until Step 6:4.0 µl 5X First-Strand Buffer0.5 µl DTT (100 mM)1.0 µl dNTPs (20 mM)5.5 µl Total Volumebine the following reagents in separate microcentrifuge tubes:For preparation of 5’-RACE-Ready cDNA For preparation of 3’-RACE-Ready cDNA1.0–10 µl RNA* 1.0–11 µl RNA*1.0 µl 5’-CDS Primer A 1.0 µl 3’-CDS Primer A0–9 µl Sterile H2O 0–10 µl Sterile H2O11 µl Total Volume 12 µl Total Volume*For the control reactions, use 1 µl of Control Mouse Heart Total RNA (1 µg/µl).3.Mix contents and spin the tubes briefly in a microcentrifuge.4.Incubate tubes at 72°C for 3 minutes, then cool the tubes to 42°C for 2 minutes. After cooling, spinthe tubes briefly for 10 seconds at 14,000 x g to collect the contents at the bottom.NOTE: This step can be performed in a thermal cycler. While the tubes are incubating, you can prepare the Master Mix in Step 6.5.To just the 5’-RACE cDNA synthesis reaction(s), add 1 µl of the SMARTer II A Oligonucleotideper reaction.6.Prepare enough of the following Master Mix for all 5’- and 3’-RACE-Ready cDNA synthesisreactions. Mix these reagents at room temperatures in the following order:5.5 µl Buffer Mix from Step 10.5 µl RNase Inhibitor (40 U/µl)2.0 µl SMARTScribe Reverse Transcriptase (100 U)8.0 µl Total Volume7.Add 8 µl of the Master Mix from Step 6 to the denatured RNA from Step 4 (3’-RACE cDNA) andStep 5 (5’-RACE cDNA), for a total volume of 20 µl per cDNA synthesis reaction.8.Mix the contents of the tubes by gently pipetting, and spin the tubes briefly to collect the contents atthe bottom.9.Incubate the tubes at 42°C for 90 minutes in an air incubator or a hot-lid thermal cycler.NOTE: Using a water bath for this incubation may reduce the volume of the reaction mixture (due to evaporation), and therefore reduce the efficiency of first-strand cDNA synthesis.10.Heat tubes at 70°C for 10 minutes.11.Dilute the first-strand cDNA synthesis reaction product with Tricine-EDTA Buffer:∙Add 10 µl if you started with <200 ng of total RNA.*∙Add 90 µl if you started with >200 ng of total RNA.*∙Add 240 µl if you started with poly A+ RNA.*The copy number of your gene of interest should be the determining factor for diluting your sample.If you have 200 ng of total RNA but your gene of interest has low abundance, dilute with 10 µl. If you have 200 ng of total RNA and the gene of interest is highly abundant, dilute with 90 µl.12.You now have 3’- and 5’-RACE-Ready cDNA samples. Samples can be stored at –20°C for up tothree months.VI. Rapid Amplification of cDNA Ends (RACE)PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.At this point, you have 3’- and 5’-RACE-Ready cDNA samples. The RACE reactions in this section use only a fraction of this material for each RNA of interest. There is sufficient single-stranded cDNA for PCR amplification of multiple genes.A. Things You Should Know Before Starting RACE PCR ReactionsIf you intend to use LD PCR to construct your full-length cDNA after completing 5’- and 3’-RACE, besure to set aside an aliquot of the 5’-RACE-Ready cDNA to use as a template in the PCR reaction.Please note that the efficiency of RACE PCR depends on the abundance of the mRNA of interest in yourRNA sample. Additionally, different primers will have different optimal annealing/extensiontemperatures. Refer to the Troubleshooting Guide (Appendix A) for suggestions on optimizing PCRconditions.NOTE: This is a RACE-specific protocol. It differs from the general SeqAmp protocol in many regards.B. Protocol: Rapid Amplification of cDNA Ends (RACE)This procedure describes the 5’-RACE and 3’-RACE PCR reactions that generate the 5’ and 3’ cDNAfragments. We recommend that you also perform positive control 5’- and 3’-RACE using the TFRprimers and UPM. Although the Universal Primer Short (UPM short) is provided, nested PCR isgenerally not necessary in SMARTer RACE reactions.1.Prepare enough PCR Master Mix for all of the PCR reactions plus one extra reaction to ensuresufficient volume. The same Master Mix can be used for both 5’- and 3’-RACE reactions. For each50 µl PCR reaction, mix the following reagents:15.5 µl PCR-Grade H2O25.0 µl 2X SeqAmp Buffer1.0 µl SeqAmp DNA Polymerase41.5 µl Total Volume2.Prepare PCR reactions as shown below in Table 2. Add the components to 0.5 ml PCR tubes in theorder shown and mix gently.Table 2. Setting up 5'- and 3'-RACE PCR ReactionsComponent 5’- or 3’-RACESampleUPM only(– control)GSP only(– control)5’- or 3’-RACE-Ready cDNA(experimental)2.5 µl 2.5 µl 2.5 µl 10X UPM 5 µl 5 µl —5’ or 3’ GSP (10 µM) 1 µl — 1 µl H2O — 1 µl 5 µl Master Mix (Step 1) 41.5 µl 41.5 µl 41.5 µl Total Volume 50 µl 50 µl 50 µl。