Cloning shRNA Oligos into pLKO.1

Resuspend oligos in ddH2O to a concentration of 20 μM.

Add 5ul Forward oligo

5

Add 5ul Reverse oligo

5

Add 5ul 10X NEB Buffer 2

5µL

Add 35 μL ddH2O

35

Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water.

0h 4m 0s

Incubate the sample at 70°C for 10 minutes in a PCR machine.

0h 10m 0s

Slowly cool to room temperature over the period of several hours.

3h 0m 0s

Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA)

6

with 5 μL 10x NEB buffer 1

5

with 1 μL AgeI

1

Bring up to 50 μl with ddH2O

Incubate at 37°C for 2 hours.

2h 0m 0s

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

5

with 1 μL EcoRI

1

and 14 μL ddH2O

14

Incubate at 37°C for 2 hours.

2h 0m 0s

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.

2

With 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above.

20

With 2 μL 10x NEB T4 DNA ligase buffer

2

With 1 μL NEB T4 DNA ligase

1

Bring up to 20ul with ddH2O

Incubate at 16°C for 4-20 hours.

4h 0m 0s

Transform 2 μL of ligation mix into 25 μL competent cells, following manufacturer’s protocol.

Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).