Jie:LabNotes/CpgSeq/2009-5-29

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Bisulfite conversion of PGP DNA for cpg30k ASM validation

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
20 GM20431 256ng/ul x 1 tube 8ul 12ul 130ul 56.1ng/ul x 10ul
21 PGP1_iPS1 50ng/ul x 1 tubes 10ul 10ul 130ul 40.7ng/ul x 10ul
22 PGP1_L 293ng/ul x 1 tubes 6.8ul 13.2ul 130ul 191.6ng/ul x 10ul
23 PGP3_L 242ng/ul x 1 tubes 6.8ul 13.2ul 130ul 114.7ng/ul x 10ul
24 PGP9_L 249ng/ul x 1 tubes 6.8ul 13.2ul 130ul 106.5ng/ul x 10ul
09_8 DF6-9-9_P16(4) 504.8ng/ul x 3 tubes 4ul 16ul 130ul 285.7ng/ul x 10ul
09_9 foreskin 583ng/ul x 2 tube 4ul 16ul 130ul 212.5ng/ul x 10ul


  • Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
  • Add 130ul of CT conversion reagent to samples.
  • 98C 10min -> 64C 150min -> 4c overnight.
  • Add 600ul M-Binding buffer to spin columns.
  • Add the converted samples to the columns, close the gap and mix by inverting several times.
  • Spin at 15,000rpm for 30sec.
  • Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
  • Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
  • Add 200ul M-Wash Buffer, spin for 30 sec.
  • Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
  • Measure the DNA with Nanodrop:RNA

set up the capture reaction

No sample sample concentration 10xLigase buffer template+cpg30k(10ng/ul,2009-05-29 by Alan)+suppressor(8nM each)vol+H2O template+v2.1(21ng/ul) vol+H2O template+v2.2(21ng/ul) vol+H2O template+v2.3(20ng/ul) vol+H2O template+Jan09#2(17ng/ul_05/20) vol+H2O
09_8 DF6-9-9_P16(4) 285.7ng/ul x 10ul 1ul 1+3+1+3ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5 2+5ul+2ul
09_9 foreskin 212.5ng/ul x 10ul 1ul 1+3+1+3ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5 2+5ul+2ul
positive control Jurkat bis-gDNA 200ng/ul 1ul 1+3+1+6ul
negative control 0 1ul 0+3+1+6ul

dNTP concentration 10mM(NEB)

                                                             ddH2O  Ligase Buffer   Ligase  Amplitaq dNTP           
2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP:  0.5ul    0.1ul    0.1ul       0.2ul    (10mM)0.1ul 
                                                          x60  30ul     6ul       6ul       12ul      6ul

PCR with AmpF/R6.2Sol and iProof

                                     x10
  Template                 5ul      
  2X iProof Mastermix     50ul      500ul
  AmpF6.2SoL (10uM)        4ul       40ul
  AmpR6.2SoL (10uM)        4ul       40ul   
  50X SYBG I             0.8ul        8ul
  H2O                   36.2ul      362ul

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. I purified the amplicons with Qiaquick. Gel purified the cpg30k four subsets amplicons and normalized the final amplicons with 1:1 ratio. The 09_8 and 09_9 cpg97k amplicons concentrations are:

09_8: 11.3ng/ul x 30ul; 09_9: 13.7ng/ul x 30ul.

==PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40

I did the dUTP_PCR with template from the mixture captured targets, the Qiaquick purified captured targets of 97k, and the directed captured targets of 97k.

reaction system                                                 x8     x6
H2O                                                42.6ul     340.8ul    
2x Master mix                                        50ul      400ul      
dUTP(1mM)                                             2ul       16ul       
AmpF6.3NH2(10uM)                                      2ul       16ul       
AmpR6.3NH2(10uM)                                      2ul       16ul     
50x SYBG I                                          0.4ul      3.2ul    
template                       0.25ul/each for cpg30k mixture, 0.05ul/each for purified cpg97k, 1ul/each for direct cpg97k.  
Total                                               100ul      1400ul     
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C

Qiaquick purification: No_09_8_30k: 34ng/ul x 60ul; No_09_8_97k: 21ng/ul x 60ul; No_09_9_30k: 36ng/ul x 60ul; No_09_8_97k: 24ng/ul x 60ul;

USER and S1 digestion

add 3ul USER to 30ul of each samples. 37C for 1h.

                                          x4 
10 x S1 nuclease buffer:  4 ul            16ul
DNA after USER digestion: 33ul            33ul each
S1 nuclease (10U/ul):      1ul            4ul
ddH2O                      2ul            8ul
37C 10mins.
Minelute cloumn purify. Elute in 16ul H2O.

end repair

                                     positive control                 x5
Total                  25ul           H2O 13ul             
DNA                    15ul            2ul(20080801_spacer_107bp)             
dNTP                  2.5ul           2.5ul                           12.5 
dATP                  2.5ul           2.5ul                           12.5
10xendrepair buffer   2.5ul           2.5ul                           12.5 
enzyme                0.5ul           0.5ul                           2.5
extra ATP(10mM)       2.5ul           2.5ul                           12.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

Egel size selection

select ~100bp fragments.

adapator ligation

                                         x6
DNA                         35ul                       
100uM Solexa_1 adaptor     0.5ul         3
100uM Solexa_2 adaptor     0.5ul         3
2xQuickLiage buffer         40ul         240
QuickLigase enzyme(NEB)      1ul         6
extra ATP(10mM)              4ul         24  
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.

Nick-translation

set up the ligation system:
                                         x11                 x5
Total                       25ul                     40
DNA                         20ul                     30    
10x ThermoPol buffer       2.5ul         27.5ul       4      20
10mM dNTP                  0.5ul          5.5ul     0.8       4
1mg/ml BSA                 2.5ul         27.5ul       4      20
Bst polymerase(8U/ul)        1ul          11ul      1.5      7.5
65C for 25 minutes -> keep on ice.

do gel size selection, harvest DNA at ~160-175bp size range.

PCR of sequencig library

                                              x11     x10
Nick-translated DNA          25ul             
Solexa_PCR_up(10uM)           2ul            22ul      200
Solexa_PCR_lo(10uM)           2ul            22ul      200
2xiProof master mix          50ul           550ul      500
50x SYBG                    0.8ul           8.8ul       8
ddH2O                        22ul           220ul      220
98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20090605 sol seq lib No09 8 9 97K.jpg20090605_sol seq lib No09_8_9_97K

Do qiaquick purification and egel cut. Gel quantification:

 No09_8_97k: 2ng/ul (15nM)
 No09_9_97k: 1.5ng/ul (11nM)