Brandon:Protocols/Scratch
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Protocol:
- 1. tagmentation with nextera kit
- 2. Partially denature at 72C (5-10C higher than the Tm for ME sequences)
- 3. Anneal a ME-T7 adaptor. make it specific for orange/blue too?
- 4. Gap filling and ligation. (T4 DNAP/Ampligase as in Shendure protocol)
- 5. IVT and conversion to sequencing libraries.
- normal tagmentation protocol (cross check with nextera tagmentation protocol)
1. tagmentation reaction (using 5 ng Jurkat DNA in reactions)
Dilute the nextera enzyme mix: 1:10 For each rxn, used mix of: 1ul 5x LMW Buffer 2ul cell lysate 1ul diluted enzyme 1ul H2O ---------------------- 5ul total / reaction 55C 10 min
2. protease digestion (to stop reaction)
To each tube, add: 1 uL 1:100 diluted Qiagen Protease (stock is 5 AU, diluted 10X, then added to tube for .5 AU/uL final []) Incubate: 50 C 10 minutes, 70 C 20 minutes
3. Partial denaturation, and addition of ME-T7 adaptor
a. add 1 uL of 10 uM ME-T7 adaptor to solution (over saturate to outcompete ME sequence?) b. incubate solution at 75C for 5 minutes (use different temps/times? see what shendure did) c. cool reaction mixture to RT at 0.1 C/s (annealing done in Shendure protocol)
3. Fill in ligation and appending of ME-T7 adaptors
- see how much T4 DNAP to add. and amp ligase, use what was used in shendure protocol)
- 3´→ 5´ exonuclease activity issue? invT on ME-T7? use BST polymerase?
- kill with protease digestion or use AMPure beads purification at this step?
4. Denature strands and anneal with T7 primer for second strand synthesis.
- use taq polymerase for fill in
- if any left over ME sequence that binds in front of the T7-sss primer will be destroyed due to the exonuclease activity of taq.
5. then can perform IVT?
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