Brandon:Protocols/Scratch: Difference between revisions

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3. Partial denaturation, and addition of ME-T7 adaptor
3. Partial denaturation, and addition of ME-T7 adaptor
   
   
  a. add 1 uL of 10 uM ME-T7 adaptor to solution  
  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)
  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)
  c. cool reaction mixture to RT at 0.1 C/s (annealing done in Shendure protocol)
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*see how much T4 DNAP to add. and amp ligase, use what was used in shendure protocol)
*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?
* 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.
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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Revision as of 07:51, 21 May 2012

leave so can always edit

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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?

*

blah