Jie:LabNotes/CpgSeq/2008-9-5

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Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit (3rd)

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
4 hFib2 68.4ng/ul x 3 tubes 20ul 0ul 130ul 34.1ng/ul x 30ul 2.1/2.7
5 hFib2_iPS4 51.9ng/ul x 3 tubes 20ul 0ul 130ul 47.4ng/ul x 30ul 2.0/2.5
6 IMB90 50ng/ul x 1 tube 20ul 0ul 130ul 36ng/ul x 10ul 1.8/2.4
14 Hues66 99ng/ul x 3 tubes 20ul 0ul 130ul 17ng/ul x 30ul 1.8/1.9
15 Hues63 207ng/ul x 3 tubes 10ul 10ul 130ul 132ng/ul x 30ul 2.2/2.7


  • 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 system

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
4 hFib2 34.1ng/ul x 30ul + 28.4ng/ul x 20ul 1ul 6+1+1+1ul 8+1.5 10+1.5 x 2 tubes 10+1.5
5 hFib2_iPS4 47.4ng/ul x 30ul + 37.8ng/ul x 20ul 1ul 6+1+1+1ul 8+1.5 10+1.5 x 2 tubes 10+1.5
6 IMB90 36ng/ul x 10ul + 22.9ng/ul x 20ul 1ul see page 08-08-15 8+1.5 10+1.5 x 2 tubes 10+1.5
14 Hues66 17ng/ul x 40ul 1ul 8+1+1 10+1.5 12+1.5 10+1.5
15 Hues63 132ng/ul x 30ul 1ul 4+1+1+3 6+1.5+1.5 10+1.5 8+1.5
Jurkat 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 see page 08-08-15 1.5+1.5+6
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 2+1.5+5.5 1.5+1.5+6


 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       0.2ul    (10mM)0.02ul 
                                                           x300       174ul           30ul        30ul        60ul      6ul
 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(09/8/2008)

                                      x5
   Template                10ul      
   2X iProof Mastermix     50ul      250ul
   AmpF6.2SoL (10uM)        4ul       20ul
   AmpR6.2SoL (10uM)        4ul       20ul   
   50X SYBG I             0.8ul        4ul
   H2O                   31.2ul      156ul
   

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

File:20080907 captured PCR N0 4 5.jpg20080907_captured PCR_N0_4_5

File:20080907 captured PCR N0 6 14.jpg20080907_captured PCR_N0_6_14

File:20080907 captured PCR N0 15 Jurkat.jpg20080907_captured PCR_N0_15_Jurkat

quantification of PCR products

yield: No_4:

        cpg30k + suppressor: 5ng/ul x 15ul
        V2.1: 5ng/ul x 15ul
        V2.2: 4ng/ul x 15ul
        V2.3: 4.5ng/ul x 15ul
        mix 3ul cpg30K + 3ul V2.1 + 4ul V2.2 + 3ul V2.3 = 5ng/ul x 13ul
      No_5:
        cpg30k + suppressor: 4ng/ul x 15ul
        V2.1: 5ng/ul x 15ul
        V2.2: 3ng/ul x 15ul
        V2.3: 3ng/ul x 15ul
        mix 3ul cpg30k + 2.5ul V2.1 + 4ul V2.2 + 4ul V2.3 = 4ng/ul x 14.5ul
      No_15:
        cpg30k + suppressor: 6ng/ul x 15ul
        V2.1: 6ng/ul x 15ul
        V2.2: 4.5ng/ul x 15ul
        V2.3: 4ng/ul x 15ul
        mix 3ul cpg30k + 3ul V2.1 + 4ul V2.2 + 4.5ul V2.3 = 4.5ng/ul x 14.5ul
      No_6: (2008-09-14)
        cpg30k + suppressor: 9ng/ul
        V2.1: 9ng/ul
        mix 4ul cpg30k + 3ul V2.1 = 9ng/ul x 7ul

sequencing library construction

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

 reaction system                                                 x4     
 H2O                                                42.6ul     170.4ul    
 2x Master mix                                        50ul      200ul      
 dUTP(1mM)                                             2ul        8ul       
 AmpF6.3(10uM)                                         2ul        8ul       
 AmpR6.3(10uM)                                         2ul        8ul       
 50x SYBG I                                          0.4ul      1.6ul       
 template(4ng/ul)                                    0.5ul        2ul each     
 Total                                               100ul      400ul      
 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Bioneer column purification. Yield:
 No_4: 52.8ng/ul x 30ul 
 No_5: 33.1ng/ul x 30ul
 No_15: 42.2ng/ul x 30ul
 No_6:                       (2008-09-14)

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.

File:200809010 dUTP-PCR MmeI No 4 5 15.jpg200809010_dUTP-PCR_MmeI_No_4_5_15

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.

File:20080912 sequencing library No 4 5 15.jpg20080912_sequencing library_No_4_5_15

Qiaquick column purification. Yield:

 No_4: hFib2, 27.1ng/ul (164nM) x 30ul
 No_5: hFib2_iPS4, 22.2ng/ul (134nM) x 30ul
 No_15: HUES_63, 30.8ng/ul (186nM) x 30ul