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


{| {{table}}
* 1:50 dilution of Nextera transposomes: 1ul Nextera enzyme in 24ul TE and 25ul 100% Glyceral
| align="center" style="background:#f0f0f0;"|'''gDNA'''
| align="center" style="background:#f0f0f0;"|'''1ul'''
|-
| HMW buffer||1ul
|-
| transposomes||1ul
|-
| Nf water||2ul
|-
| Total ||5ul
|}


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

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