Rui:Haplotying: Difference between revisions
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Add Proteinase right before aliquot, 1ul per tube, to 20 tubes. Store at -80C. | Add Proteinase right before aliquot, 1ul per tube, to 20 tubes. Store at -80C. | ||
Step 3: Tagmentation | 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== | ==Primers== |
Revision as of 23:26, 16 March 2011
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>