ShRNA Cloning
Oligo Design
[edit]to be added
Cloning
[edit]Annealing Step
[edit]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.
Vector Digestion
[edit]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
Ligation
[edit]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
Transformation
[edit]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.
Screening
[edit]1. Make miniprep, digest with restriction enzymes.
2. Sequencing.