Brandon:Protocols/Scratch

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Revision as of 08:08, 17 May 2012 by >Bsos (Created page with "==leave so can always edit== ===blah=== Protocol: 1. tagmentation with nextera kit 2. Partially denature at 72C (5-10C higher than the Tm for ME sequences) 3. Anneal a ME-...")
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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 enzyme: 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. Partial denaturation, and addition of ME-T7 adaptor

a. add 1 uL of 10 uM ME-T7 adaptor to solution 
b. incubate solution at 72C for 5 minutes
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)


4. Denature strands and anneal with T7 primer for second strand synthesis.





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