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

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===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 ===
===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 ===
   
   
   reaction system                                                x6      
   reaction system                                                x14     x2
   H2O                                                42.6ul    255.6ul    
   H2O                                                42.6ul    596.4ul    
   2x Master mix                                        50ul      300ul      
   2x Master mix                                        50ul      700ul      
   dUTP(1mM)                                            2ul      12ul        
   dUTP(1mM)                                            2ul      28ul        
   AmpF6.3(10uM)                                        2ul      12ul        
   AmpF6.3(10uM)                                        2ul      28ul        
   AmpR6.3(10uM)                                        2ul      12ul      
   AmpR6.3(10uM)                                        2ul      28ul      
   50x SYBG I                                          0.4ul      2.4ul    
   50x SYBG I                                          0.4ul      5.6ul    
   template(4ng/ul)                                    0.5ul      2 uleach     
   template(4ng/ul)                                    0.5ul      2 uleach     
   Total                                              100ul      600ul      
   Total                                              100ul      1400ul      


   94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
   94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C


   Bioneer column purification. Yield:
   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===
===Digestion with MmeI===
                                       No_4_5_15    x3
                                       No_21~26      x6
   Total                                30ul                  
   Total                                40ul                  
   dUTP_PCR                            20ul         
   dUTP_PCR                            20ul         
   10X NEBuffer 4                      4ul          12            
   10X NEBuffer 4                      4ul          24            
   1mM SAM(fresh)                      4ul         12              
   1mM SAM(fresh)                      8ul         48              
   2U/ul Mme I                          8ul          24                
   2U/ul Mme I                          8ul          48                
  ddH2O                                4ul          12
 
 
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
   37C 2h. MinEulte column purification. Elute in 12ul EB.
   37C 2h. MinEulte column purification. Elute in 12ul EB.
Line 160: Line 167:


===step4. S1 nuclease digestion===
===step4. S1 nuclease digestion===
                             No4_5_15      x3
                             No21~26        x7
   10 x S1 nuclease buffer:  2ul            6ul
   10 x S1 nuclease buffer:  2ul            14ul
   DNA after USER digestion: 13ul            13ul
   DNA after USER digestion: 13ul            13ul
   S1 nuclease (10U/ul):      1ul            3ul
   S1 nuclease (10U/ul):      1ul            7ul
   ddH2O                      4ul            12ul
   ddH2O                      4ul            28ul


   37C 10mins.
   37C 10mins.
Line 170: Line 177:


===step5. end repair===
===step5. end repair===
                       No4_5_15         positive control               x4
                       No21~26         positive control                 x7
   Total                  25ul          H2O 13ul             
   Total                  25ul          H2O 13ul             
   DNA                    15ul            2ul(20080801_spacer_107bp)             
   DNA                    15ul            2ul(20080801_spacer_107bp)             
   dNTP                  2.5ul          2.5ul                          10
   dNTP                  2.5ul          2.5ul                          17.5
   dATP                  2.5ul          2.5ul                          10
   dATP                  2.5ul          2.5ul                          17.5
   10xendrepair buffer  2.5ul          2.5ul                          10
   10xendrepair buffer  2.5ul          2.5ul                          17.5
   enzyme                0.5ul          0.5ul                           2
   enzyme                0.5ul          0.5ul                           3.5
   extra ATP(10mM)      2.5ul          2.5ul                          10
   extra ATP(10mM)      2.5ul          2.5ul                          17.5


   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
   Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
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===step6. adapter ligation===
===step6. adapter ligation===
                            
                            
                           No4_5_15   positive control    negative control        x5
                           No21~26   positive control    negative control        x8
   DNA                        13ul          13ul            ddH2O 13ul                 
   DNA                        13ul          13ul            ddH2O 13ul                 
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul              2.5
   100uM Solexa_1 adaptor    0.5ul        0.5ul                0.5ul              4
   100uM Solexa_2 adaptor     0.5ul        0.5ul                0.5ul              2.5
   100uM Solexa_2_PE adaptor 0.5ul        0.5ul                0.5ul              4
   2xQuickLiage buffer        15ul          15ul                  15ul              75
   2xQuickLiage buffer        15ul          15ul                  15ul              120
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul               5
   QuickLigase enzyme(NEB)      1ul          1ul                  1ul             8
   extra ATP(10mM)            2.5ul        2.5ul                2.5ul              12.5  
   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.
   Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.
Line 197: Line 204:
    
    
   set up the ligation system:
   set up the ligation system:
                                           x5              
                                           x8              
   Total                      20ul               
   Total                      20ul               
   DNA                        15ul               
   DNA                        15ul               
   10x ThermoPol buffer        2ul        10ul
   10x ThermoPol buffer        2ul        16ul
   10mM dNTP                  0.4ul         2ul     
   10mM dNTP                  0.4ul         3.2ul     
   1mg/ml BSA                  2ul        10ul    
   1mg/ml BSA                  2ul        16ul    
   Bst polymerase(8U/ul)        1ul          5ul    
   Bst polymerase(8U/ul)        1ul          8ul    


   65C for 25 minutes -> keep on ice.
   65C for 25 minutes -> keep on ice.


===step8. PCR of sequencig library===
===step8. PCR of sequencig library===
                                                 x5
                                                 x8
   Nick-translated DNA          20ul             
   Nick-translated DNA          20ul             
   Solexa_PCR_up(10uM)          2ul            10ul  
   Solexa_PCR_up(10uM)          2ul            16ul  
   Solexa_PCR_lo(10uM)           2ul            10ul
   Solexa_PCR_PE_loH(10uM)       2ul            16ul
   2xiProof master mix          50ul          250ul  
   2xiProof master mix          50ul          400ul  
   50x SYBG                    0.8ul             4ul
   50x SYBG                    0.8ul           6.4ul
   ddH2O                      25.2ul          126ul
   ddH2O                      25.2ul          201.6ul
   98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
   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;