Jie:LabNotes/CpgSeq/2009-1-22

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

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

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)

                                      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

 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:
 

Digestion with MmeI

                                     No_4_5_15     x3 
 Total                                30ul                  
 dUTP_PCR                             20ul         
 10X NEBuffer 4                       4ul          12           
 1mM SAM(fresh)                       4ul          12             
 2U/ul Mme I                          8ul          24               
 ddH2O                                4ul          12
 
 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

                            No4_5_15      x3 
 10 x S1 nuclease buffer:   2ul            6ul
 DNA after USER digestion: 13ul            13ul
 S1 nuclease (10U/ul):      1ul            3ul
 ddH2O                      4ul            12ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair

                      No4_5_15         positive control               x4
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                           10 
 dATP                  2.5ul           2.5ul                           10
 10xendrepair buffer   2.5ul           2.5ul                           10 
 enzyme                0.5ul           0.5ul                            2
 extra ATP(10mM)       2.5ul           2.5ul                           10
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation

                          No4_5_15    positive control     negative control        x5
 DNA                         13ul          13ul            ddH2O 13ul                
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul              2.5
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul              2.5
 2xQuickLiage buffer         15ul          15ul                  15ul              75
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul               5
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul              12.5  
 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:
                                          x5              
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul         10ul 
 10mM dNTP                  0.4ul          2ul    
 1mg/ml BSA                   2ul         10ul   
 Bst polymerase(8U/ul)        1ul          5ul    
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library

                                               x5
 Nick-translated DNA          20ul             
 Solexa_PCR_up(10uM)           2ul            10ul  
 Solexa_PCR_lo(10uM)           2ul            10ul
 2xiProof master mix          50ul           250ul  
 50x SYBG                    0.8ul             4ul
 ddH2O                      25.2ul           126ul
 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.