Rui:Haplotying
Jump to navigation
Jump to search
Background
Original: File:Design.ppt
Modified: File:Tn-based-haplotyping V1.1.ppt
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 7.5AU)
Step 2: Proteinase and aliquot
Add Proteinase right before aliquot, 1ul per tube, to 20 tubes. Store at -80C.
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 10ul (dNTP;polymerase) 0.5uM T7 adaptor 1ul 100* SYBR green 0.25ul Nf water up to 50ul Total 50ul
Primers
File:Primers for Tn-based-haplotyping.xls
2011
<calendar> name=Rui:LabNotes/Haplotyping format=%name/%year-%month-%day date=2011/10/1 view=oneyear </calendar>