Cloning shRNA Oligos into pLKO.1
Resuspend oligos in ddH2O to a concentration of 20 μM.
Add 5ul Forward oligo. 
Add 5ul Reverse oligo. 
Add 5 μL	10x NEB buffer 2. 
Add 35 μL ddH2O. 
Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water.
Incubate the sample at 70°C for 10 minutes in a PCR machine.
Slowly cool to room temperature over the period of several hours.
Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) with 5 μL	10x NEB buffer 1 with 1 μL	AgeI bring to 50 μL	ddH2O. 
Incubate at 37°C for 2 hours.
Purify with Qiaquick gel extraction kit, eluting in 30 μL of ddH2O.
Digest eluate with EcoRI by mixing: 30 μL pLKO.1 TRC-cloning vector digested with AgeI with 5 μL	10x NEB buffer for EcoRI with 1 μL	EcoRI and 14 μL	ddH2O. 
Incubate at 37°C for 2 hours.
Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb.
Cut out the 7kb band and place in a sterile microcentrifuge tube.
Purify the DNA using a Qiaquick gel extraction kit.
Elute in 30 μL of ddH2O.
Measure the DNA concentration.
Use your ligation method of choice.
For a standard T4 ligation, mix: 2 μL	annealed oligo from "Annealing Oligos" section above.
With 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above.
With 2 μL	10x NEB T4 DNA ligase buffer With 1 μL	NEB T4 DNA ligase Bring up to 20ul with ddH2O. 
Incubate at 16°C for 4-20 hours.
Transform 2 μL of ligation mix into 25 μL competent DH5 alpha cells, following manufacturer’s protocol.
Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).
