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

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   reaction system                                                x6     
   reaction system                                                x6     
   H2O                                                42.6ul    170.4ul    
   H2O                                                42.6ul    255.6ul    
   2x Master mix                                        50ul      200ul      
   2x Master mix                                        50ul      300ul      
   dUTP(1mM)                                            2ul       8ul        
   dUTP(1mM)                                            2ul       12ul        
   AmpF6.3(10uM)                                        2ul       8ul        
   AmpF6.3(10uM)                                        2ul       12ul        
   AmpR6.3(10uM)                                        2ul       8ul        
   AmpR6.3(10uM)                                        2ul      12ul   
   50x SYBG I                                          0.4ul      1.6ul     
   50x SYBG I                                          0.4ul      2.4ul   
   template(4ng/ul)                                    0.5ul       2ul each      
   template(4ng/ul)                                    0.5ul       2 uleach      
   Total                                              100ul      400ul     
   Total                                              100ul      600ul   


   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

Revision as of 22:07, 28 January 2009

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                                                 x6     
 H2O                                                42.6ul     255.6ul    
 2x Master mix                                        50ul      300ul      
 dUTP(1mM)                                             2ul       12ul       
 AmpF6.3(10uM)                                         2ul       12ul       
 AmpR6.3(10uM)                                         2ul       12ul     
 50x SYBG I                                          0.4ul      2.4ul    
 template(4ng/ul)                                    0.5ul       2 uleach     
 Total                                               100ul      600ul     
 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.