lenticrispr-cas9-protocol
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Lenticrispr-cas9 knockout genes in cell lines
Modified by yongtang 20140701
1.lenticrispr-cas9 vector digestion
vector: 4ug
10X buffer 3.1 5ul
BsmbI 1ul
ddH2OXul
total 50ul
55℃for 1h. then add 1ul CIP , 37℃for 1h.
2.If BsmBI digested, a ~2kb filler piece should be present on the gel. Only gel purify the large (~11kb) band. Leave the 2kb band.
3.gRNA anneal
oligo 1 (1ug/ml) 10ul
oligo 2 (1ug/ml) 10ul
5X forward buffer 6ul
total 26ul
95℃denature for 5 min, then cool down on block to room temperature.
4.gRNA phosphorylation
annealgRNA 8ul
10mM ATP 1ul
10U/ul T4 kinase 1ul
total 10ul
37℃for 60min, then 65℃for 10min.
5.ligate and transform
lenticrispr-cas9 vector (bsmbI digest) 30ng
gRNA (anneal and phosphorylation) 1ng or 6ng
10X ligation buffer 1ul
T4 ligase (400u/ul) 1ul
BSA (1mg/ml) 1ul
total 10ul
16℃ligate overnight. Then transform into DH5a.
6.select the positive clone for sequence.
7.Lenti-virus packaging
For 10cm dish:
Add 2X HBSS, then mix well;
Add CaCl2 drop by drop, then slowly mix the solution;
Wait for 30 min, at room temperature;
Change medium after 10-12h, then harvest after 72h;
Collect medium from 293FT gently, spin 3000rpm for 10min and then collect the medium;
Spin the medium 20,000g for 2h at 4℃, then leave the medium;
Resuspended the pellet with 300ul medium (without FBS).
8.Infect cell
Infect cell (in 6 well plate), every well for 300ul virus medium and 8ug/ml polybrane. After 12h ,change the medium. After 48h, add the puromycin to the medium for selecting positive cells( the puromycinconcentration depends on your cell line).
9.Western blot
After 7-14 days , collect some cells for western blot confirm.