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

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>Sam Chiang
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>Sam Chiang
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===MmeI digestion===
===MmeI digestion===
  Digestion with MmeI
  Digestion with MmeI
                                                 x7
                                                 x2
  Total                                50ul                  
  Total                                80ul                  
  PCR                                  28ul          
  PCR                                  48ul          
  10X NEBuffer 4                        5ul       35            
  10X NEBuffer 4                        8ul       16            
  1mM SAM(fresh)                      10ul       70              
  1mM SAM(fresh)                      16ul       32              
  2U/ul Mme I                          8ul        56                
  2U/ul Mme I                          8ul        16                


  1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 50ul EB.
  1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 50ul EB.
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===endrepair with enzymatic end-repair kit===
===endrepair with enzymatic end-repair kit===


                                       x8
                                       x2
  28 ul DNA  
  28 ul DNA  
  4 ul 10X End-Repair Buffer          32
  4 ul 10X End-Repair Buffer          8
  4 ul dNTP Mix                        32
  4 ul dNTP Mix                        8
  4 ul End-Repair Enzyme Mix          32
  4 ul End-Repair Enzyme Mix          8
  40 ul Total reaction volume
  40 ul Total reaction volume
  Qiaquick column purification. Elute in 30ul EB.
  Qiaquick column purification. Elute in 30ul EB.
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===A tail addition===
===A tail addition===


                                         x8
                                         x2
  Blunt-ended DNA                30ul    30 each
  Blunt-ended DNA                30ul    30 each
  10X Klenow buffer              4ul    32
  10X Klenow buffer              4ul    8
  1mM dATP                        6ul    48
  1mM dATP                        6ul    12
  Klenow fragment (exo-)10U/ul    2ul    16
  Klenow fragment (exo-)10U/ul    2ul    4
  37C 30min, purified with MinElute columns, eluted with 12ul EB.  
  37C 30min, purified with MinElute columns, eluted with 12ul EB.  



Revision as of 16:34, 29 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
                                                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.

endrepair with enzymatic end-repair kit

                                     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
Qiaquick column purification. Elute in 30ul EB.

A tail addition

                                        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

                                             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: 175~300bp.