Rui:Haplotying

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Revision as of 23:26, 16 March 2011 by >RuiLiu (→‎Protocol)
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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>