Jie:LabNotes/CpgSeq/2009-9-28: Difference between revisions

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==Exom-samples Sequencing library construction without shearing==
==Exom-samples Sequencing library construction without shearing==


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


===Pool the products together===
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===
===PCR with Amp6.3NH2 primer===
  I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
  I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
  reaction system                                                x4      x6  
  reaction system                                                x4      x2  
  H2O                                                  45ul      180ul    1080
  H2O                                                  45ul      180ul    360
  2x iProof Mastermix                                  50ul      200ul    1200
  2x iProof Mastermix                                  50ul      200ul    400
  AmpF6.3NH2(10uM)                                      2ul      8ul      48
  AmpF6.3NH2(10uM)                                      2ul      8ul      16
  AmpR6.3NH2(10uM)                                      2ul      8ul      48
  AmpR6.3NH2(10uM)                                      2ul      8ul      16
  50x SYBG I                                          0.4ul      1.6ul    9.6
  50x SYBG I                                          0.4ul      1.6ul    3.2
  template                                      1ul/each for purified amplicons
  template                                      1ul/each for purified amplicons
  Total                                              100ul      800ul     
  Total                                              100ul      800ul     

Revision as of 23:24, 28 September 2009

Exom-samples Sequencing library construction without shearing

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

Pool the products together

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

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

Digestion with MmeI
                                                x7 
Total                                50ul                  
PCR                                  28ul         
10X NEBuffer 4                        5ul        35           
1mM SAM(fresh)                       10ul        70             
2U/ul Mme I                           8ul        56               
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 30ul EB.