Jie:LabNotes/CpgSeq/2009-9-8

From ZhangLabWiki
Jump to navigation Jump to search

shotgun library construction of PGP samples and new batch DFiPS captured by cpg97k[edit]

The library construction started with the Covaris sheared DNA: 
The captured DNA of PGP samples were sent to Billy for shearing.refer to LabNotes on [1] 
The captured DNA of new batch DFiPS samples were sent to Billy for shearing.refer to LabNotes on [2] 

digest PGP samples with MmeI[edit]

                                                  x7 
Total                               160ul      
DNA                                 100ul            
10X NEBuffer 4                       16ul         112
1mM SAM(fresh)                       32ul         224
2U/ul Mme I                           8ul          56 
ddH2O                                4ul           28

1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
37C 2h
MinElute column purify. Elute in 20ul EB. 

endrepair with enzymatic end-repair kit[edit]

PGP samples                            x7
 20 ul DNA 
  3 ul 10X End-Repair Buffer           21
  3 ul dNTP Mix                        21
  4 ul End-Repair Enzyme Mix           28
  30 ul Total reaction volume
DF-iPS                                 x2
100 ul DNA 
13 ul 10X End-Repair Buffer            26
13 ul dNTP Mix                         26
 4 ul End-Repair Enzyme Mix            8
130 ul Total reaction volume
Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O.
E-gel size selection:
P09_2,P09_3,DF30k and DF97K: select 100bp;
The others: select 150bp.

A tail addition[edit]

                                          x9
  Blunt-ended DNA                30ul     30 each
  10X Klenow buffer               4ul     36
  1mM dATP                        6ul     54
  Klenow fragment (exo-)10U/ul    2ul     18
  37C 30min, purified with MinElute columns, eluted with 12ul EB. 

adaptor ligation[edit]

                                               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 Minelute columns, eluted with 10ul EB. 
    TBU gel size selection: 175~300bp.
File:20090909 TBU size selection.jpg20090909_TBU size selection

PCR[edit]

   Template               15ul        
   2x iProof mix          50ul       
   Solexa_PCR_up (10uM)    4ul        
   Solexa_PCR_lo_PE (10uM) 4ul        
   H2O                    27ul       
   50X SYBG I             0.2ul
98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 72C 3min -> 15C hold.
Purify with qiaquick column and size selection with e-gel.


quantification of shotgun library with qPCR[edit]

I dilute the PhiX lib(10nM) to 1nM, 0.25nM, 0.05nM, 0.01nM.
I dilute the sample libraries with 1:10 ratio. 
Syb_FP5: ATGATACGGCGACCACCGAG
Syb_RP7: CAAGCAGAAGACGGCATACGAG
                                      x 24
  Template                0.5ul
  2x iProof mix           12.5ul       
  syb_RP7 (100uM)        0.1ul        
  Syb_FP5 (100uM)        0.1ul        
  H2O                     12ul       
  50X SYBG I             0.1ul
98C 30sec -> 30 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.
File:20090910 shotgun library quantification.png20090910_shotgun library quantification_control
Media:20090910_shotgun library quantification.xls20090910_shotgun library quantification_excel table
No sample sample concentration
P09_1 1.56nM
P09_2 4735 3.12nM
P09_3 4789 5.3nM
P09_4 5171 3.06nM
P09_5 1818 nM
P09_6 1947 nM
P09_7 1741 nM
DFiPS_30k 4977 8.58nM
DFiPS_97k 4879 8.72nM
Alice sequenced P09_4(NA21660*1 053007) and mixture of DFiPS30K and DFiPS97K (8.65nM) on 09/10/09.