ShRNA Cloning

Oligo Design

to be added

In PCR tube, mix the following:
1 ul 10X PCR buffer (no Triton X-100, no DMSO)

2 ul sense (top) oligo (10 uM)
2 ul antisense (bottom) oligo (10uM)

5 ul water to 10 final volume.

Incubate the tube for 2 minutes at 95°C.

Turn off PCR machine immediately and let the tube cool down to RT.

Store the annealed oligos at 4 oC before ligation or store at -20 °C for long term storage.

Digest vector with restriction enzymes, gel purify vector, elute with 15 ul water.

Note: make sure you use fresh 0.5X TAE and wash electrophoresis tank before running a gel

Add

5 ul annealed oligo mix to gel-purified vector.

2 ul 10X ligation buffer or 15 ul 2X fast ligation buffer

0.5 ul T4 DNA ligase, or T7 DNA ligase

Incubate at RT for 1 ~ 2 hours or O/N

Add 50 ul STBL3 or Top10 competent cells to 10 ul ligation mix.

Incubate for 30 min on ice.

Heat shock at 42 oC for 1 min, cool on ice for 2 min.

Add 300 LB/Y or recovery medium and incubate at 37 oC (shaking, 750 rpm) for 30 min to 1 hour.

Spin down cells, plate on LB/Amp or LB/Kan plate.

1. Make miniprep, digest with restriction enzymes.

2. Sequencing.