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: Proteinase digestion
Incubate at 50C, 15min.
Reference link: [http://www.qiagen.com/products/accessories/accessoryenzymes/qiagenprotease.aspx?ShowInfo=1]


Step 3: gDNA Tagmentation
Step 3: gDNA Tagmentation

Revision as of 21:55, 29 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: Proteinase digestion

Incubate at 50C, 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

<calendar> name=Rui:LabNotes/Haplotyping format=%name/%year-%month-%day date=2011/10/1 view=oneyear </calendar>