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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