Jie:LabNotes/CpgSeq/2009-9-28

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Exom-samples Sequencing library construction without shearing[edit]

I constructed the library with captured product found under Alice's labnotes 8/15/09 and 8/16/09

Pool the products together[edit]

pooled all the subsets according ALice's labnote on 8/20/09
                        CV-ips 1      CV-ips 2     CV-ips 3     CV-ips 4        
concnetration(ng/ul):  31.3ng/ul     10.2ng/ul    25ng/ul      12ng/ul
volume needed (ul):    2ul           3ul          1.8ul        3.8ul
--------------------------------------------------------------------------
total (ng):           62.6ng         31.3ng       46ng         45ng
                       CV-fb 1        CV-fb 2      CV-fb 3      CV-fb 4       
concnetration(ng/ul): 12.3ng/ul       6.1ng/ul     11.7ng/ul    11.2ng/ul
volume needed (ul):   3ul             3ul          2.3ul        2.4ul
--------------------------------------------------------------------------
total (ng):           36.9ng          18.5ng       27ng         26.5ng


PCR with Amp6.3NH2 primer[edit]

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4       x2  
H2O                                                  45ul      180ul     360
2x iProof Mastermix                                  50ul      200ul     400 
AmpF6.3NH2(10uM)                                      2ul       8ul      16 
AmpR6.3NH2(10uM)                                      2ul       8ul      16
50x SYBG I                                          0.4ul      1.6ul     3.2
template                                      1ul/each for purified amplicons
Total                                               100ul      800ul     
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->72C 3min.
Purify with Qiaquick column. Elute in 100ul EB.

MmeI digestion[edit]

Digestion with MmeI
                                                x2 
Total                                80ul                  
PCR                                  48ul         
10X NEBuffer 4                        8ul        16           
1mM SAM(fresh)                       16ul        32             
2U/ul Mme I                           8ul        16               
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 50ul EB.
28ul samples are used for endrepair and 22ul are used for ligation directly.
File:20090928 after MmeI.jpg20090928_after MmeI

endrepair with enzymatic end-repair kit[edit]

                                     x2
28 ul DNA 
4 ul 10X End-Repair Buffer           8
4 ul dNTP Mix                        8
4 ul End-Repair Enzyme Mix           8
40 ul Total reaction volume
RT for 30 min.
Qiaquick column purification. Elute in 30ul EB.

A tail addition[edit]

                                        x2
Blunt-ended DNA                30ul     30 each
10X Klenow buffer               4ul     8
1mM dATP                        6ul     12
Klenow fragment (exo-)10U/ul    2ul     4
37C 30min, purified with MinElute columns, eluted with 12ul EB. 

adaptor ligation[edit]

                                             x8
  DNA                                10ul
  2x QuickLigase buffer (enzymatic)  15ul     120
  20uM Adaptor oligo mix              3ul      24
  T4 DNA QuickLigase (enzymatic)      2ul      16
  Incubate at RT for 15 minutes.
  Purified with Minelute columns, eluted with 10ul EB. 
  TBU gel size selection: 200bp, 225-275bp, >275bp.

PCR[edit]

File:20090930 lib.jpg20090930_lib

quantification with Q-PCR[edit]

CViPS: 27.37nM;
CVfibroblast: 19.6nM.