Jie:LabNotes/CpgSeq/2009-8-5: Difference between revisions

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==shotgun library construction of Parkinson's samples==
==shotgun library construction of Parkinson's samples==
====endrepair with enzymatic end-repair kit====
The captured DNA of samples were sent to Covaris for shearing.refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-7-1] see 1.5.2
===endrepair with enzymatic end-repair kit===
                                         x10
                                         x10
  100 ul DNA  
  100 ul DNA  
Line 9: Line 10:
  Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.
  Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.


==== A tail addition====
=== A tail addition===
                                     x11
                                     x11
   Blunt-ended DNA        10ul      10 each
   Blunt-ended DNA        10ul      10 each
Line 17: Line 18:
   37C 30min, purified with MinElute columns, eluted with 12ul EB.  
   37C 30min, purified with MinElute columns, eluted with 12ul EB.  


====adaptor ligation====
===adaptor ligation===
                                                 x12
                                                 x12
     DNA                                10ul
     DNA                                10ul
Line 26: Line 27:
     Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O.
     Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O.


====PCR====
===PCR===


     Template                5ul         
     Template                5ul         
Line 36: Line 37:
  98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)->  
  98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)->  
  72C 3min -> 15C hold.
  72C 3min -> 15C hold.
[[Image:20090807_shotgun_lib_Parkinson_samples.jpg]]20090807_shotgun_lib_Parkinson_samples

Revision as of 20:59, 10 August 2009

shotgun library construction of Parkinson's samples

The captured DNA of samples were sent to Covaris for shearing.refer to LabNotes on [1] see 1.5.2

endrepair with enzymatic end-repair kit

                                       x10
100 ul DNA 
 13 ul 10X End-Repair Buffer           130
 13 ul dNTP Mix                        130
  4 ul End-Repair Enzyme Mix            40
130 ul Total reaction volume
Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.

A tail addition

                                   x11
  Blunt-ended DNA        10ul      10 each
  10X Klenow buffer      1.6ul     17.6
  1mM dATP                3ul      33
  Klenow fragment (exo-)  1ul      11
  37C 30min, purified with MinElute columns, eluted with 12ul EB. 

adaptor ligation

                                               x12
    DNA                                10ul
    2x QuickLigase buffer (enzymatic)  15ul     180
    20uM Adaptor oligo mix              3ul      36
    T4 DNA QuickLigase (enzymatic)      2ul      24
    Incubate at RT for 15 minutes.
    Purified with Qiaquick columns, eluted with 12ul EB. Do the TBU gel size selection of 200~225bp fragments. ethanol precipitation and elute in 10ul ddH2O.

PCR

   Template                5ul        
   2x iProof mix          50ul       
   Solexa_PCR_up (10uM)    4ul        
   Solexa_PCR_lo_PE (10uM) 4ul        
   H2O                     37ul       
   50X SYBG I             0.2ul
98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)-> 
72C 3min -> 15C hold.

File:20090807 shotgun lib Parkinson samples.jpg20090807_shotgun_lib_Parkinson_samples