Brandon:Protocols/Scratch

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Revision as of 23:35, 20 May 2012 by >Bsos (→‎blah)
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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 
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?

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

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