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
 1ul DNA or cell lysate (can use 5 ng/ul jurkat DNA)
 2ul 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: 50C 10 minutes, 70C 20 minutes
(reaction 6 uL total)

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
c. cool reaction mixture to 45C at 0.1 C/s (annealing done in Shendure protocol)
d. leave at 45C for 10 minutes, then cool to 37C at 0.1 C/s
e. let sit at 37C for 10 minutes.
(reaction 7 uL total)

3. Fill in ligation and appending of ME-T7 adaptors

  • buffer already present from tagmentation is compatible with buffers for ampligase and T4 DNA polymerase
transposome reaction mixture
5× Tagmentation reaction buffer 
50 mM Tris-OAc pH 8.0
25 mM Mg(OAc)2

ampligase buffer comp
2X Quick Ligation Reaction Buffer: 
132 mM Tris-HCL 
20 mM MgCl2 
2 mM dithiothreitol 
2 mM ATP 
15% Polyethylene glycol (PEG 6000)  
pH 7.6 @25°C

T4 DNAP buffer comp
1X T4 DNA Ligase Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM Dithiothreitol
pH 7.5 @ 25°C


  • shendure 3μl of Ampligase at 5U/μl (Epicentre – Illumina) and 1μl of either T4 DNAP
  • 3´→ 5´ exonuclease activity issue? invT on ME-T7? use BST polymerase?
  • kill with protease digestion or use AMPure beads purification at this step?
  • (reaction 11 uL total)

4. Fill in reaction (second strand synthesis)

  • couple PCR cycles to ensure

Add 6 uL 2X taq polymerase, run at 72C for 10 minutes. (nextera does 3 mins? longer templates?) IVT on filled in reaction and not filled in reaction in to show fill-in is working.

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

  • 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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