【工具】CRISPR sgRNA Oligo設計

張峰的好多sgRNA vector用的restriction site 都是BsmBI,所以這裡用BsmBI 舉例。

找到BsmBI site，圈住，如下圖。

BsmBI site的開頭是CACC,結尾是CAAA

設計oligo的時候要確保在開頭的基礎上加G，以確保穩定性。

所以設計Oligo的時候是：

• Forward：5 - CACCG - 20bp - 3
• Reverse：5- AAAC -20bp（Copy bottom strand 5->3） - C - 3 (注意後面還跟一個C！！！)

Oligo粘合之後，應該長成下圖這樣：

實驗步驟：

1. digest Vector:
1. 20ug vector + 20ul NEB buffer 3.1 + 10ul BsmBI +H2O =200ul
2. 55C, 4-6h
2. purify vector:
1. prepare 1% gel, 40ul 6x loading dye into to 200ul
2. load into wells, with undigested vector as control
3. 120v, 3hs
4. extract DNA, isopropanol precipitation(optional)
5. DNA in 2000ul TE (6ng/ul)
6. freeze the opened vector (for current and future use)
3. Anneal oligo:
1. 100uM oligos in H2O
2. Mix: 1.5ul Forward +1.5ul Reverse + 5ul NEB 3.1 +42ul H2O = 50ul
3. 95C, 4min
4. to make sure it cool very slowly(important!), get 70C water into 1L beaker, incubate the tube in the water, let the water cool to RT (few hours), then its ready
4. Ligation:
1. 1ul annealed oligos / 1ul water as control
2. 3ul opened vector
3. 2ul 10x ligase buffer
4. 13ul H2O
5. 1ul T4 ligase
6. mix, 16C, 3-4h
5. Transformation into E.coli:
1. 2ul ligation into 25 ul Ecoli, 30min ice
2. 42C, 30-90s (depend on which strain)
3. 400ul SOC, 37C shake 1h
4. 150ul to Amp plate
5. 37C, overnight
6. pick 2-4 colonies, into LB + Amp
7. 37C for less than 17h
8. stock glycerol: 150ul Glycerol + 850ul (15% glycerol) in -80C
6. Sequencing to see if sgRNA is there:
1. primer is on U6 promoter: 5- GATACAAGGCTGTTAGAGAGATAATT - 3

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