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 ) .
