TITLE: Cloning shRNA Oligos into pLKO.1
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol accompanying the "pLKO.1 – TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: http://www.addgene.org/tools/protocols/plko/</div></div>

[STEPS]
?. [Annealing Oligos]
Resuspend oligos in ddH2O to a concentration of 20 μM.
?. [Annealing Oligos]
Add 5ul Forward oligo
5 µl
?. [Annealing Oligos]
Add 5ul Reverse oligo
5 µl
?. [Annealing Oligos]
Add 5 μL	10x NEB buffer 2
5 µl
?. [Annealing Oligos]
Add 35 μL ddH2O
35 µl
?. [Annealing Oligos]
Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water.
?. [Annealing Oligos]
Incubate the sample at 70°C for 10 minutes in a PCR machine.
?. [Annealing Oligos]
Slowly cool to room temperature over the period of several hours.
This will take a few hours, but it is important for the cooling to occur slowly for the oligos to anneal.
If using a beaker of water, remove the beaker from the flame, and allow the water to cool to room temperature.
?. [Digesting pLKO.1 TRC Cloning Vector]
Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA)
6 µg
?. [Digesting pLKO.1 TRC Cloning Vector]
with 5 μL	10x NEB buffer 1
5 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
with 1 μL	AgeI
1 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
bring to 50 μL	ddH2O
50 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
Incubate at 37°C for 2 hours.
?. [Digesting pLKO.1 TRC Cloning Vector]
Purify with Qiaquick gel extraction kit, eluting in 30 μL of ddH2O.
?. [Digesting pLKO.1 TRC Cloning Vector]
Digest eluate with EcoRI by mixing: 30 μL pLKO.1 TRC-cloning vector digested with AgeI
?. [Digesting pLKO.1 TRC Cloning Vector]
with 5 μL	10x NEB buffer for EcoRI
5 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
with 1 μL	EcoRI
1 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
and 14 μL	ddH2O
14 µl
?. [Digesting pLKO.1 TRC Cloning Vector]
Incubate at 37°C for 2 hours.
?. [Digesting pLKO.1 TRC Cloning Vector]
Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb.
When visualizing DNA fragments to be used for ligation, use only long-wavelength UV light. Short wavelength UV light will increase the chance of damaging the DNA.
?. [Digesting pLKO.1 TRC Cloning Vector]
Cut out the 7kb band and place in a sterile microcentrifuge tube.
?. [Digesting pLKO.1 TRC Cloning Vector]
Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.
?. [Digesting pLKO.1 TRC Cloning Vector]
Measure the DNA concentration.
?. [Ligating and Transforming into Bacteria]
Use your ligation method of choice. For a standard T4 ligation, mix: 2 μL	annealed oligo from "Annealing Oligos" section above.
2 µl
?. [Ligating and Transforming into Bacteria]
With 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above.
20 ng
If you were unable to measure the DNA concentration, use 1 μL
?. [Ligating and Transforming into Bacteria]
With 2 μL	10x NEB T4 DNA ligase buffer
2 µl
?. [Ligating and Transforming into Bacteria]
With 1 μL	NEB T4 DNA ligase
1 µl
?. [Ligating and Transforming into Bacteria]
Bring up to 20ul with ddH2O
?. [Ligating and Transforming into Bacteria]
Incubate at 16°C for 4-20 hours.
?. [Ligating and Transforming into Bacteria]
Transform 2 μL of ligation mix into 25 μL competent DH5 alpha cells, following manufacturer’s protocol.
?. [Ligating and Transforming into Bacteria]
Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).