Rui:Haplotying
Background
Original: File:Design.ppt
Modified: File:Tn-based-haplotyping V1.1.ppt
Human gDNA samples: Kun's stack - the box at the left up corner (-20C), Rui's box (4C) with GC DNA and Africa's DNA.
Protocol
Step 1: Cell lysis
Collect 10 cells in 20ul lysis buffer.
Cell lysis buffer (1*) 200mM NaCl 20mM EDTA 10mM Tris-HCl (pH 8) 0.2% TritonX-100 (0.1 AU/ml Qiagen Proteinase stock:7.5AU/2ml)
Step 2: Proteinase and aliquot
Add Proteinase right before aliquot, 1ul per tube, to 20 tubes. Store at -80C.
Step 3: Proteinase digestion
Incubate at 50C, 15min.
Inactivation at 70C, 15min
Reference link: [1]
Step 3: gDNA Tagmentation
- 1:50 dilution of Nextera transposomes: 1ul Nextera enzyme in 24ul TE and 25ul 100% Glyceral
gDNA 1ul 5X HMW buffer 1ul transposomes 1ul Nf water 2ul Total 5ul
55C for 5min
Quick spin and cool down on ice
Step 4: Addition of T7 adaptor by Kapa system (?)
Tagmented gDNA 5ul Kapa master mix 1X (dNTP;polymerase) 0.5uM T7 adaptor 1ul 100* SYBR green 0.25ul Nf water up to 50ul Total 50ul
72C, 3min; 95C, 30sec
95C, 10sec; 62C, 30sec; 72C, 3min; 5 cycles
Step 5: ExoI (SAP) treatment (?)
Removal of T7 adaptor is a must for IVT, maybe optional for PCR as the following step...
10U/ul ExoI mixed with 1U/ul SAP as 1.5~5U:1U, add 2ul (?) for each tube
37C, 30min; 80C, 15-25min
Step 6: Mix and EtOH precipitation in 2ml tube
Mix 10 tubes (~500ul) for EtOH precipitation
Glycoblue 2ul NaOAc 50ul EtOH 1250ul Total 1800ul
-80C for 1hr; wash with 70% and air-dry (optional); resuspend in 10ul water
Step 7: PCR or IVT
PCR:
barcode mixed gDNA 5ul 2* Nextera PCR buffer 25ul 50* primer cocktail 1ul Nextera Enzyme 1ul 100* SYBR green 0.25ul Nf water 17.75ul Total 50ul
IVT with ABi kit:
barcode mixed gDNA 5ul IVT buffer 1ul IVT master mix 3ul IVT Enzyme mix 1ul Enhancer 1ul Total 11ul
Primers
File:Primers for Tn-based-haplotyping.xls
2011
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