Jie:LabNotes/CpgSeq/2009-1-22: Difference between revisions

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{| border="1" cellpadding="5" cellspacing="0" align="center"
{| border="1" cellpadding="5" cellspacing="0" align="center"
|-
|-
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|'''No'''
| align="center" style="background:#f0f0f0;"|'''sample '''
| align="center" style="background:#f0f0f0;"|'''sample '''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
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| align="center" style="background:#f0f0f0;"|'''260:280/260:230'''
| align="center" style="background:#f0f0f0;"|'''260:280/260:230'''
|-
|-
|21||PGP1_iPS1||50ng/ul x 3 tubes||20ul||0ul||130ul||ng/ul x 10ul||2.18/2.97
|21||PGP1_iPS1||50ng/ul x 3 tubes||20ul||0ul||130ul||179.1ng/ul x 10ul||1.97/1.72
|-
|-
|22||PGP1_L||293ng/ul x 3 tubes||6.8ul||13.2ul||130ul||ng/ul x 20ul||2.19/2.91
|22||PGP1_L||293ng/ul x 3 tubes||6.8ul||13.2ul||130ul||368.7ng/ul x 20ul||2.13/2.04
|-
|-
|23||PGP3_L||242ng/ul x 3 tubes||6.8ul||13.2ul||130ul||ng/ul x 20ul||2.05/2.78
|23||PGP3_L||242ng/ul x 3 tubes||6.8ul||13.2ul||130ul||238.6ng/ul x 20ul||2.07/1.89
|-
|-
|24||PGP9_L||249ng/ul x 3 tubes||6.8ul||13.2ul||130ul||ng/ul x 20ul||2.3/3.0
|24||PGP9_L||249ng/ul x 3 tubes||6.8ul||13.2ul||130ul||223.7ng/ul x 20ul||2.05/1.89
|-
|-
|25||PGP1F.2||25ng/ul x 4 tubes||20ul||0ul||130ul||ng/ul x 10ul||1.37/1.01
|25||PGP1F.2||25ng/ul x 4 tubes||20ul||0ul||130ul||84ng/ul x 10ul||1.67/1.2
|-
|-
|26||PGP9F.2||14ng/ul x 4 tube||20ul||0ul||130ul||ng/ul x 10ul||
|26||PGP9F.2||14ng/ul x 4 tube||20ul||0ul||130ul||35.1ng/ul x 10ul||1.2/0.66
|-
|-
|}
|}
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RNA
RNA


==set up the capture system==
==set up the capture reaction==


{| border="1" cellpadding="5" cellspacing="0" align="center"
{| border="1" cellpadding="5" cellspacing="0" align="center"
|-
|-
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|'''No '''
| align="center" style="background:#f0f0f0;"|'''sample '''
| align="center" style="background:#f0f0f0;"|'''sample '''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
|align="center" style="background:#f0f0f0;"|'''sample concentration'''
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| align="center" style="background:#f0f0f0;"|'''template+v2.3(20ng/ul) vol+H2O'''
| align="center" style="background:#f0f0f0;"|'''template+v2.3(20ng/ul) vol+H2O'''
|-
|-
|1||BJ||43.3ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5
|21||PGP1_iPS1||179.1ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
|-
|2||BJ_iPS_11||65.6ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5
|22||PGP1_L||368.7ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
|-
|3||BJ-iPS_12||43.3ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5
|23||PGP3_L||238.6ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
|-
|6||IMB90||22.9ng/ul x 30ul||1ul||4+1+1+3ul|| || ||  
|24||PGP9_L||223.7ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
|-
|9||Hybrid_2||174.5ng/ul x 10ul + 65.6 x 10ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5
|25||PGP1F.2||84ng/ul x 10ul||1ul||7+1+1+0ul||||||
|-
|-
| ||positive control||200ng/ul||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6
|26||PGP9F.2||35.1ng/ul x 10ul||1ul||7+1+1+0ul||||||
|-
|-
| ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6
| ||positive control||200ng/ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|-
| ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5
|}
|}




   dNTP concentration 10mM(NEB)
   dNTP concentration 10mM(NEB)
                                                                      ddH2O     Ligase Buffer  Ligase     Amplitaq   dNTP           
                                                                    ddH2O Ligase Buffer  Ligase   Amplitaq   dNTP           
   2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP:     0.58ul         0.1ul       0.1ul       0.2ul    (10mM)0.02ul  
   2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP:   0.58ul     0.1ul     0.1ul     0.2    (10mM)0.02ul  
                                                             x300       174ul          30ul        30ul        60ul     6ul
                                                             x100    58ul       10ul      10ul    20ul     2ul


   95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
   95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.


==PCR(08/16/2008)==
==PCR(01/26/2009)==


                                       x8
                                       x12
     Template                10ul       
     Template                10ul       
     2X iProof Mastermix    50ul      400ul
     2X iProof Mastermix    50ul      600ul
     AmpF6.2SoL (10uM)        4ul      32ul
     AmpF6.2SoL (10uM)        4ul      48ul
     AmpR6.2SoL (10uM)        4ul      32ul    
     AmpR6.2SoL (10uM)        4ul      48ul    
     50X SYBG I            0.8ul      6.4ul
     50X SYBG I            0.8ul      9.6ul
     H2O                  31.2ul    249.6ul
     H2O                  31.2ul    374.4ul
      
      
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
  [[Image:20090126_No_21_22.jpg]]20090126_No_21_22
  [[Image:20090126_No_23_24_25_26.jpg]]20090126_No_23_24_25_26
==quantification of gel purified amplicon==
  no_21 PGP1_iPS1: cpg30k 1ng/ul x 10ul;
                  V2.1 1ng/ul x 10ul;
                 
  No_22 PGP1_L: cpg30k 3ng/ul x 10ul;
  No_23 PGP3_L: cpg30k 3ng/ul x 10ul;
                V2.1 3 ng/ul x 10ul;
                V2.2 1ng/ul x 10ul;
                V2.3 3ng/ul x 10ul;
  No_24 PGP9_L: cpg30k 3ng/ul x 10ul;
                V2.1 3 ng/ul x 10ul;
                V2.3 3ng/ul  x 10ul;
  No_25 PGP1F.2: cpg30k 2ng/ul;
  No_26 PGP9F.2: cpg30k 2ng/ul;
  no_21 PGP1_iPS1: cpg30k 1ng/ul 2ul + V2.1 1ng/ul x 2ul;
  No_22 PGP1_L: cpg30k 3ng/ul x 2ul;
  No_23 PGP3_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.2 1ng/ulx 2ul + V2.3 3ng/ul x 1ul;
  No_24 PGP9_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.3 3ng/ul x 1ul;
  No_25 PGP1F.2: cpg30k 2ng/ul x 2ul;
  No_26 PGP9F.2: cpg30k 2ng/ul x 2ul;
==sequencing library construction==
===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 ===
  reaction system                                                x14    x2
  H2O                                                42.6ul    596.4ul   
  2x Master mix                                        50ul      700ul     
  dUTP(1mM)                                            2ul      28ul     
  AmpF6.3(10uM)                                        2ul      28ul     
  AmpR6.3(10uM)                                        2ul      28ul   
  50x SYBG I                                          0.4ul      5.6ul   
  template(4ng/ul)                                    0.5ul      2 uleach   
  Total                                              100ul      1400ul   
  94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
  Bioneer column purification. Yield:
  No_21:40.5ng/ul;
  No_22:39.1ng/ul;
  No_23:35.6ng/ul;
  No_24:32.4ng/ul;
  No_25:47ng/ul;
  No_26:35.2ng/ul;
  No_21_2: 28.9ng/ul;
===Digestion with MmeI===
                                      No_21~26      x6
  Total                                40ul                 
  dUTP_PCR                            20ul       
  10X NEBuffer 4                      4ul          24         
  1mM SAM(fresh)                      8ul          48           
  2U/ul Mme I                          8ul          48             
 
  1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
  37C 2h. MinEulte column purification. Elute in 12ul EB.
===step3. USER digestion===
  DNA                    10ul       
  USER                    3ul       
  total                  13ul         
37C 2h.
===step4. S1 nuclease digestion===
                            No21~26        x7
  10 x S1 nuclease buffer:  2ul            14ul
  DNA after USER digestion: 13ul            13ul
  S1 nuclease (10U/ul):      1ul            7ul
  ddH2O                      4ul            28ul
  37C 10mins.
  Minelute cloumn purify. Elute in 16ul H2O.
===step5. end repair===
                      No21~26        positive control                x7
  Total                  25ul          H2O 13ul           
  DNA                    15ul            2ul(20080801_spacer_107bp)           
  dNTP                  2.5ul          2.5ul                          17.5
  dATP                  2.5ul          2.5ul                          17.5
  10xendrepair buffer  2.5ul          2.5ul                          17.5
  enzyme                0.5ul          0.5ul                          3.5
  extra ATP(10mM)      2.5ul          2.5ul                          17.5
  Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
===step6. adapter ligation===
                         
                          No21~26    positive control    negative control        x8
  DNA                        13ul          13ul            ddH2O 13ul               
  100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul              4
  100uM Solexa_2_PE adaptor  0.5ul        0.5ul                0.5ul              4
  2xQuickLiage buffer        15ul          15ul                  15ul              120
  QuickLigase enzyme(NEB)      1ul          1ul                  1ul              8
  extra ATP(10mM)            2.5ul        2.5ul                2.5ul              20 
  Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.
  TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.
===step7. Nick-translation===
 
  set up the ligation system:
                                          x8             
  Total                      20ul             
  DNA                        15ul             
  10x ThermoPol buffer        2ul        16ul
  10mM dNTP                  0.4ul        3.2ul   
  1mg/ml BSA                  2ul        16ul 
  Bst polymerase(8U/ul)        1ul          8ul   
  65C for 25 minutes -> keep on ice.
===step8. PCR of sequencig library===
                                                x8
  Nick-translated DNA          20ul           
  Solexa_PCR_up(10uM)          2ul            16ul 
  Solexa_PCR_PE_loH(10uM)      2ul            16ul
  2xiProof master mix          50ul          400ul 
  50x SYBG                    0.8ul          6.4ul
  ddH2O                      25.2ul          201.6ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
[[Image:20090130_parallel Solexa Sequencing library No21~26.jpg]]20090130_parallel Solexa Sequencing library No21~26
  Qiaquick column purification and quantification:
  No_21 PGP1_iPS1: 16.4 ng/ul(90.3nM) x 30ul;
  No_22 PGP1_L: 14.1 ng/ul(77.6nM) x 30ul;
  No_23 PGP3_L: 14.7 ng/ul(80.9nM) x 30ul;
  No_24 PGP9_L: 17.6 ng/ul(96.9nM) x 30ul;
  No_25 PGP1_F.2: 18.5 ng/ul(93.4nM) x 30ul;
  No_26 PGP9_F.2: 19.3 ng/ul(97.4nM) x 30ul;

Latest revision as of 00:39, 31 January 2009

6th Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit[edit]

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn 260:280/260:230
21 PGP1_iPS1 50ng/ul x 3 tubes 20ul 0ul 130ul 179.1ng/ul x 10ul 1.97/1.72
22 PGP1_L 293ng/ul x 3 tubes 6.8ul 13.2ul 130ul 368.7ng/ul x 20ul 2.13/2.04
23 PGP3_L 242ng/ul x 3 tubes 6.8ul 13.2ul 130ul 238.6ng/ul x 20ul 2.07/1.89
24 PGP9_L 249ng/ul x 3 tubes 6.8ul 13.2ul 130ul 223.7ng/ul x 20ul 2.05/1.89
25 PGP1F.2 25ng/ul x 4 tubes 20ul 0ul 130ul 84ng/ul x 10ul 1.67/1.2
26 PGP9F.2 14ng/ul x 4 tube 20ul 0ul 130ul 35.1ng/ul x 10ul 1.2/0.66


  • 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[edit]

No sample sample concentration 10xLigase buffer template+cpg30k(50.7ng/ul)+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
21 PGP1_iPS1 179.1ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
22 PGP1_L 368.7ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
23 PGP3_L 238.6ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
24 PGP9_L 223.7ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
25 PGP1F.2 84ng/ul x 10ul 1ul 7+1+1+0ul
26 PGP9F.2 35.1ng/ul x 10ul 1ul 7+1+1+0ul
positive control 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5


 dNTP concentration 10mM(NEB)
                                                                   ddH2O  Ligase Buffer   Ligase   Amplitaq   dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP:   0.58ul      0.1ul     0.1ul     0.2     (10mM)0.02ul 
                                                           x100     58ul       10ul       10ul     20ul      2ul
 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> 
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.

PCR(01/26/2009)[edit]

                                      x12
   Template                10ul      
   2X iProof Mastermix     50ul      600ul
   AmpF6.2SoL (10uM)        4ul       48ul
   AmpR6.2SoL (10uM)        4ul       48ul   
   50X SYBG I             0.8ul      9.6ul
   H2O                   31.2ul    374.4ul
   

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.

 File:20090126 No 21 22.jpg20090126_No_21_22
 File:20090126 No 23 24 25 26.jpg20090126_No_23_24_25_26

quantification of gel purified amplicon[edit]

 no_21 PGP1_iPS1: cpg30k 1ng/ul x 10ul;
                  V2.1 1ng/ul x 10ul;
                  
 No_22 PGP1_L: cpg30k 3ng/ul x 10ul;
 No_23 PGP3_L: cpg30k 3ng/ul x 10ul;
               V2.1 3 ng/ul x 10ul;
               V2.2 1ng/ul x 10ul;
               V2.3 3ng/ul x 10ul;
 No_24 PGP9_L: cpg30k 3ng/ul x 10ul;
               V2.1 3 ng/ul x 10ul;
               V2.3 3ng/ul  x 10ul;
 No_25 PGP1F.2: cpg30k 2ng/ul;
 No_26 PGP9F.2: cpg30k 2ng/ul;
 no_21 PGP1_iPS1: cpg30k 1ng/ul 2ul + V2.1 1ng/ul x 2ul;
 No_22 PGP1_L: cpg30k 3ng/ul x 2ul;
 No_23 PGP3_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.2 1ng/ulx 2ul + V2.3 3ng/ul x 1ul;
 No_24 PGP9_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.3 3ng/ul x 1ul;
 No_25 PGP1F.2: cpg30k 2ng/ul x 2ul;
 No_26 PGP9F.2: cpg30k 2ng/ul x 2ul;

sequencing library construction[edit]

2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40[edit]

 reaction system                                                 x14     x2
 H2O                                                42.6ul     596.4ul    
 2x Master mix                                        50ul      700ul      
 dUTP(1mM)                                             2ul       28ul       
 AmpF6.3(10uM)                                         2ul       28ul       
 AmpR6.3(10uM)                                         2ul       28ul     
 50x SYBG I                                          0.4ul      5.6ul    
 template(4ng/ul)                                    0.5ul       2 uleach     
 Total                                               100ul      1400ul     
 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Bioneer column purification. Yield:
 No_21:40.5ng/ul;
 No_22:39.1ng/ul;
 No_23:35.6ng/ul;
 No_24:32.4ng/ul;
 No_25:47ng/ul;
 No_26:35.2ng/ul;
 No_21_2: 28.9ng/ul;


Digestion with MmeI[edit]

                                     No_21~26      x6 
 Total                                40ul                  
 dUTP_PCR                             20ul         
 10X NEBuffer 4                       4ul          24           
 1mM SAM(fresh)                       8ul          48             
 2U/ul Mme I                          8ul          48               
  
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h. MinEulte column purification. Elute in 12ul EB.

step3. USER digestion[edit]

 DNA                    10ul         
 USER                    3ul        
 total                  13ul           

37C 2h.

step4. S1 nuclease digestion[edit]

                            No21~26         x7 
 10 x S1 nuclease buffer:   2ul            14ul
 DNA after USER digestion: 13ul            13ul
 S1 nuclease (10U/ul):      1ul            7ul
 ddH2O                      4ul            28ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair[edit]

                      No21~26         positive control                 x7
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                           17.5 
 dATP                  2.5ul           2.5ul                           17.5
 10xendrepair buffer   2.5ul           2.5ul                           17.5 
 enzyme                0.5ul           0.5ul                           3.5
 extra ATP(10mM)       2.5ul           2.5ul                           17.5
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation[edit]

                          No21~26    positive control     negative control        x8
 DNA                         13ul          13ul            ddH2O 13ul                
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul              4
 100uM Solexa_2_PE adaptor  0.5ul         0.5ul                 0.5ul              4
 2xQuickLiage buffer         15ul          15ul                  15ul              120
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul              8
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul              20  
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.
 TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.

step7. Nick-translation[edit]

 set up the ligation system:
                                          x8              
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul         16ul 
 10mM dNTP                  0.4ul         3.2ul    
 1mg/ml BSA                   2ul         16ul   
 Bst polymerase(8U/ul)        1ul          8ul    
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library[edit]

                                               x8
 Nick-translated DNA          20ul             
 Solexa_PCR_up(10uM)           2ul            16ul  
 Solexa_PCR_PE_loH(10uM)       2ul            16ul
 2xiProof master mix          50ul           400ul  
 50x SYBG                    0.8ul           6.4ul
 ddH2O                      25.2ul           201.6ul
 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20090130 parallel Solexa Sequencing library No21~26.jpg20090130_parallel Solexa Sequencing library No21~26

 Qiaquick column purification and quantification:
 No_21 PGP1_iPS1: 16.4 ng/ul(90.3nM) x 30ul;
 No_22 PGP1_L: 14.1 ng/ul(77.6nM) x 30ul;
 No_23 PGP3_L: 14.7 ng/ul(80.9nM) x 30ul;
 No_24 PGP9_L: 17.6 ng/ul(96.9nM) x 30ul;
 No_25 PGP1_F.2: 18.5 ng/ul(93.4nM) x 30ul;
 No_26 PGP9_F.2: 19.3 ng/ul(97.4nM) x 30ul;