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Protocols > pLKO.1 Protocol
pLKO.1 - TRC Cloning Vector
Addgene Plasmid 10878. Protocol Version 1.0. December 2006.
Copyright Addgene 2006, All Rights Reserved. This protocol is provided for your
convenience. See warranty information in appendix.
Click here for a printable copy.
Table of Contents
• A. pLKO.1-TRC Cloning Vector
o A.1 The RNAi Consortium
o A.2 Map of pLKO.1
o A.3 Related plasmids
• B. Designing shRNA Oligos for pLKO.1
o B.1 Determine the optimal 21-mer targets in your gene
o B.2 Order oligos compatible with pLKO.1
• C. Cloning shRNA oligos into pLKO.1
o C.1 Recommended materials
o C.2 Annealing oligos
o C.3 Digesting pLKO.1 TRC-Cloning Vector
o C.4 Ligating and transforming into bacteria
• D. Screening for Inserts
o D.1 Recommended materials
o D.2 Screening for inserts Plasmid Cart
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• E. Producing Lentiviral Particles
o E.1 Recommended materials
o E.2 Protocol for producing lentiviral particles
• F. Infecting Target Cells
o F.1 Recommended materials
o F.2 Determining the optimal puromycin concentration
o F.3 Protocol for lentiviral infection and selection
• G. Safety
• H. References
o H.1 Published articles
o H.2 Web resources
• I. Appendix
o I.1 Sequence of pLKO.1 TRC-Cloning Vector
o I.2 Recipes
o I.3 Warranty information
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A. pLKO.1-TRC Cloning Vector
A.1 The RNAi Consortium
The pLKO.1 cloning vector is the backbone upon which The RNAi
Consortium (TRC) has built a library of shRNAs directed against 15,000
human and 15,000 mouse genes. Addgene is working with the TRC to
make this shRNA cloning vector available to the scientific community.
Please cite Moffat et al., Cell 2006 Mar; 124(6):1283-98 (PubMed) in all
publications arising from the use of this vector.
A.2 Map of pLKO.1
pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC
for expression of shRNAs. pLKO.1 can be introduced into cells via direct
transfection, or can be converted into lentiviral particles for subsequent
infection of a target cell line. Once introduced, the puromycin resistance
marker encoded in pLKO.1 allows for convenient stable selection.
Figure 1 : Map of pLKO.1 containing an shRNA insert. The original
pLKO.1-TRC cloning vector has a 1.9kb stuffer that is released by
digestion with AgeI and EcoRI. shRNA oligos are cloned into the AgeI
and EcoRI sites in place of the stuffer. The AgeI site is destroyed in
most cases (depending on the target sequence), while the EcoRI site is
preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer,
visit /10878.
Description Vector Element
U6 Human U6 promoter drives RNA Polymerase III
transcription for generation of shRNA transcripts.
cPPT Central polypurine tract, cPPT, improves transduction
efficiency by facilitating nuclear import of the vector's
preintegration complex in the transduced cells.
hPGK Human phosphoglycerate kinase promoter drives
expression of puromycin.
Puro R Puromycin resistance gene for selection of pLKO.1
plasmid in mammalian cells.
sin 3'LTR 3' Self-inactivating long terminal repeat. f1 ori f1 bacterial origin of replication.
Amp R Ampicillin resistance gene for selection of pLKO.1 plasmid
in bacterial cells
pUC ori pUC bacterial origin of replication.
5'LTR 5' long terminal repeat.
RRE Rev response element.
Figure 2 : Detail of shRNA insert. The U6 promoter directs RNA
Polymerase III transcription of the shRNA. The shRNA contains 21
"sense" bases that are identical to the target gene, a loop
containing an XhoI restriction site, and 21 "antisense" bases that
are complementary to the "sense" bases. The shRNA is followed
by a polyT termination sequence for RNA Polymerase III.
A.3 Related Products
The following plasmids available from Addgene are recommended for use
in conjunction with the pLKO.1 TRC-cloning vector.
Plasmid (Addgene ID #) Description
pLKO.1 - TRC control
(10879) Negative control vector containing
non-hairpin insert.
pLKO.1 - scramble shRNA
(1864) Negative control vector containing
scrambled shRNA.
psPAX2 (12260) Packaging plasmid for producing viral
particles.
