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

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>Jie deng
(New page: ==Exom-samples Sequencing library construction without shearing==)
 
>Jie deng
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==Exom-samples Sequencing library construction without shearing==
==Exom-samples Sequencing library construction without shearing==
===PCR with Amp6.3NH2 primer===
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                x4      x6 
H2O                                                  45ul      180ul    1080
2x iProof Mastermix                                  50ul      200ul    1200
AmpF6.3NH2(10uM)                                      2ul      8ul      48
AmpR6.3NH2(10uM)                                      2ul      8ul      48
50x SYBG I                                          0.4ul      1.6ul    9.6
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.

Revision as of 23:22, 28 September 2009

Exom-samples Sequencing library construction without shearing

PCR with Amp6.3NH2 primer

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4       x6  
H2O                                                  45ul      180ul     1080
2x iProof Mastermix                                  50ul      200ul     1200 
AmpF6.3NH2(10uM)                                      2ul       8ul      48 
AmpR6.3NH2(10uM)                                      2ul       8ul      48
50x SYBG I                                          0.4ul      1.6ul     9.6
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.