Rui:Haplotying: Difference between revisions

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==Background==


* "Methylation haplotyping on few cells" project: May 27th email
[http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/Haplotyping/2011-5-12]
* Haplotyping on diluted gDNA (~1pg) from limited cells (~5)
Human gDNA samples: Kun's stack - the box at the left up corner (-20C), Rui's box (4C) with GC DNA and Africa's DNA.
==Protocol==
Step 1: Cell lysis
* 1X TritonX-100 lysis buffer: 10mM Tris-HCl (pH 8), 200mM NaCl, 20mM EDTA, 0.2% TritonX-100, (0.1 AU/ml Qiagen Proteinase stock:7.5AU/2ml)
* 10X NP40 lysis buffer [100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004]
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.
Inactivation at 70C, 15min
Reference link: [http://www.qiagen.com/products/accessories/accessoryenzymes/qiagenprotease.aspx?ShowInfo=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>

Latest revision as of 00:56, 7 December 2011