Jie:LabNotes/CpgSeq/2009-8-5

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Revision as of 18:40, 4 August 2009 by >Jie deng (New page: ==shotgun library construction of Parkinson's samples== ====endrepair with epicentre kit==== 100 ul DNA 15 ul 10X End-Repair Buffer 15 ul dNT...)
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shotgun library construction of Parkinson's samples

endrepair with epicentre kit

100 ul DNA 
 15 ul 10X End-Repair Buffer
 15 ul dNTP Mix
 15 ul ATP
  3 ul End-Repair Enzyme Mix
150 ul Total reaction volume
Incubate at room temperature for 45 minutes. Purify with minelute column.

A tail addition

                                   x8
  Blunt-ended DNA        10ul      80
  10X Klenow buffer      1.5ul     12
  1mM dATP                3ul      24
  Klenow fragment (exo-)  1ul       8
  37C 30min, purified with MinElute columns, eluted with 20ul EB. 
After A tail, I did size selection for No7 and positive control, then do the adaptor ligation. 
After adaptor ligation, i goes to PCR directly without size selection again.
However, I can only see the positive control product only.
So I skipped the size selection before adaptor ligation for No 1~6, and do the size selection after adaptor ligation.  
However, the expected band after PCR is so weak that I am not sure the targets were successfully amplified.

adaptor ligation

    DNA                                20ul
    10x QuickLigase buffer (enzymatic)  3ul
    20uM Adaptor oligo mix              3ul
    T4 DNA QuickLigase (enzymatic)      2ul
    Incubate at RT for 15 minutes.
    Purified with Qiaquick columns, eluted with 12ul EB

PCR

   Template               15ul        
   2x iProof mix          50ul       
   Solexa_PCR_up (10uM)    4ul        
   Solexa_PCR_lo_PE (10uM) 4ul        
   H2O                     30ul       
   50X SYBG I             0.2ul      

File:20090721 shotgun libr par7 positive control.jpg20090721_shotgun_libr_par7_positive control

File:20090721 shotgun libr par1~6.jpg20090721_shotgun_libr_par1~6

redo the shotgun library starting from PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-22)

I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample.
reaction system                                                 x4       x17  
H2O                                                  50ul      200ul     3400
2x Mastermix                                         50ul      200ul     3400 
AmpF6.3NH2(100uM)                                    0.2ul     0.8ul     13.6 
AmpR6.3NH2(100uM)                                    0.2ul     0.8ul     13.6
50x SYBG I                                          0.4ul      1.6ul     27.2
template                                      1ul/each for purified cpg97k
Total                                               100ul      800ul     
94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold.
I did the PCR reamplification with Amp6.3 primers with Parkinson's sample 1 and 10 with the same PCR condition.