Jie:LabNotes/CpgSeq/2008-8-15

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2rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit[edit]

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn 260:280/260:230
1 BJ 178ng/ul x 2 tubes 11.2ul 8.8ul 130ul 43.3ng/ul x 20ul 2.18/2.97
2 BJ_iPS_11 140ng/ul x 2 tubes 14.3ul 5.7ul 130ul 65.6ng/ul x 20ul 2.19/2.91
6 IMB90 50ng/ul 20ul 0ul 130ul 22.7ng/ul x 10ul 2.05/2.78
9 Hybrid2 200ng/ul 10ul 10ul 130ul 174.5ng/ul x 10ul 2.3/3.0
12 Hues8 178.5ng/ul 13ul 7ul 130ul 8ng/ul x 10ul 1.37/1.01
13 Hues42 20ng/ul x 2 tube 20ul 0ul 130ul 14ng/ul x 20ul


  • 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[edit]

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
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
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
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
6 IMB90 22.9ng/ul x 30ul 1ul 4+1+1+3ul
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
positive control 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 2+1.5+5.5 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(08/16/2008)[edit]

                                      x8
   Template                10ul      
   2X iProof Mastermix     50ul      400ul
   AmpF6.2SoL (10uM)        4ul       32ul
   AmpR6.2SoL (10uM)        4ul       32ul   
   50X SYBG I             0.8ul      6.4ul
   H2O                   31.2ul    249.6ul
   

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

File:20080817 PCR No1 No2.jpg20080817_PCR_No1_No2

File:20080817 PCR No3 No9.jpg20080817_PCR_No3_No9

since the cpg30kV2.2 were not amplified, I redo the bisulfite conversion and capture of cpg30kV2.2.

3rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit(2008-08-29)[edit]

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
1 BJ 178ng/ul 11.2ul 8.8ul 130ul 48.1ng/ul x 10ul
2 BJ_iPS_11 140ng/ul 14.3ul 5.7ul 130ul 74.8ng/ul x 10ul
3 BJ_iPS_12 40ng/ul 20ul 0ul 130ul 41.4ng/ul x 10ul
9 Hybrid2 200ng/ul 10ul 10ul 130ul 122.1ng/ul x 10ul
Jurkat 154ng/ul x 2 tube 10ul 10ul 130ul 201.4ng/ul x 20ul
  • 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[edit]

sample sample concentration 10xLigase buffer template+v2.2(21ng/ul) vol+H2O
1 BJ 48.1ng/ul x 10ul 1.5ul 10+1.5+2.5
2 BJ_iPS_11 74.8ng/ul x 10ul 1.5ul 10+1.5+2.5
3 BJ-iPS_12 41.4ng/ul x 10ul 1.5ul 10+1.5+2.5
9 Hybrid_2 122.1ng/ul x 10ul 1.5ul 10+1.5+2.5
positive control_Jurkat 201.4ng/ul 1.5ul 2+1.5+5.5
negative control 0 1.5ul 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       0.2ul    (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(09/01/2008)[edit]

                                      x6
   Template                10ul      
   2X iProof Mastermix     50ul      300ul
   AmpF6.2SoL (10uM)        4ul       24ul
   AmpR6.2SoL (10uM)        4ul       24ul   
   50X SYBG I             0.8ul      4.8ul
   H2O                   31.2ul    187.2ul
   

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

File:20080901 cpg30kV2.2 captured PCR No 1 2 3 9 Jurkat.jpg20080902_cpg30kV2.2_captured PCR_No1_2_3_9_Jurkat

File:20080901 Quantification of No1 2.jpg20080901_quantification of No_1_2

File:20080901 quantification of No 3 9.jpg20080901_quantificatio of No3_9

yield: No_1:
        cpg30k + suppressor: 6ng/ul x 15ul
        V2.1: 6ng/ul x 15ul
        V2.2: 4ng/ul x 15ul
        V2.3: 6ng/ul x 15ul
        V2.2_09/01/08: 3ng/ul x 15ul
        mix 5ul cpg30k + suppressor + 5ul V2.1 + 10ul V2.2_09/01/08 + 5ul V2.3 = 4.8ng/ul x 25ul         
        No_2: 
        cpg30k + suppressor: 6ng/ul x 15ul
        V2.1: 6ng/ul x 15ul
        V2.2: 3ng/ul x 15ul
        V2.3: 5ng/ul x 15ul
        V2.2_09/01/08: 4ng/ul x 15ul
        mix 5ul cpg30k + suppressor + 5ul V2.1 + 7.5ul V2.2_09/01/08 + 6ul V2.3 = 5ng/ul x 23.5ul

No_3:

        cpg30k + suppressor: 4.5ng/ul x 15ul
        V2.1: 4.5ng/ul x 15ul
        V2.2: 2.5ng/ul x 15ul
        V2.3: 2.5ng/ul x 15ul
        V2.2_09/01/08: 2ng/ul x 15ul
        mix 5ul cpg30k + suppressor + 5ul V2.1 + 4ul V2.2 + 9ulV2.3 + 5ul V2.2_09/01/08 = 3.2ng/ul x 28ul         

No_9:

        cpg30k + suppressor: 5ng/ul x 15ul
        V2.1: 3ng/ul x 15ul
        V2.2: 1.5ng/ul x 15ul
        V2.3: 2ng/ul x 15ul
        V2.2_09/01/08: 3ng/ul x 15ul
        mix 4ul cpg30k + suppressor + 7ul V2.1 + 10ul V2.3 + 7ul V2.2_09/01/08 = 2.8ng/ul x 28ul         


sequencing library construction[edit]

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

 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     
 Total                                               100ul      400ul      
 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
 Bioneer column purification. Yield:
 No_1: 33.2ng/ul x 30ul 
 No_2: 39.4ng/ul x 30ul
 No_3: 56.7ng/ul x 30ul
 No_9: 49.4ng/ul x 30ul

Digestion with MmeI[edit]

                                     No_1_2_3_9    x4 
 Total                                30ul                  
 dUTP_PCR                             20ul         
 10X NEBuffer 4                       4ul          16           
 1mM SAM(fresh)                       4ul          16             
 2U/ul Mme I                          8ul          32               
 ddH2O                                4ul          16
 
 1mM SAM: 32mM SAM 1ul + 31ul ddH2O.
 37C 2h. MinEulte column purification. Elute in 12ul EB.

File:20080901 dUTP PCR and MmeI.jpg20080901_dUTP_PCR and MmeI

step3. USER digestion[edit]

 DNA                    10ul         
 USER                    3ul        
 total                  13ul           

37C 12h.

step4. S1 nuclease digestion[edit]

                            No1_2_3_9      x4 
 10 x S1 nuclease buffer:   2ul            8ul
 DNA after USER digestion: 13ul            13ul
 S1 nuclease (10U/ul):      1ul            4ul
 ddH2O                      4ul            16ul
 37C 10mins.
 Minelute cloumn purify. Elute in 16ul H2O.

step5. end repair[edit]

                      No1_2_3_9         positive control               x5
 Total                  25ul           H2O 13ul             
 DNA                    15ul            2ul(20080801_spacer_107bp)             
 dNTP                  2.5ul           2.5ul                           12.5 
 dATP                  2.5ul           2.5ul                           12.5
 10xendrepair buffer   2.5ul           2.5ul                           12.5 
 enzyme                0.5ul           0.5ul                            2.5
 extra ATP(10mM)       2.5ul           2.5ul                           12.5
 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.

step6. adapter ligation[edit]

                          No1_2_3_9    positive control     negative control       x6
 DNA                         13ul          13ul            ddH2O 13ul              48  
 100uM Solexa_1 adaptor     0.5ul         0.5ul                 0.5ul               3
 100uM Solexa_2 adaptor     0.5ul         0.5ul                 0.5ul               3
 2xQuickLiage buffer         15ul          15ul                  15ul              90
 QuickLigase enzyme(NEB)      1ul           1ul                   1ul               6
 extra ATP(10mM)            2.5ul         2.5ul                 2.5ul              15  
 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O.
 TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.

File:20080902 gul size selectin after adaptor ligation.jpg20080902_gel size selection after adaptor ligation

File:20080902 positive and negative control of adaptor ligation.jpg20080902_gel of control and genative control

step7. Nick-translation[edit]

 set up the ligation system:
                                          x6              
 Total                       20ul              
 DNA                         15ul               
 10x ThermoPol buffer         2ul         12ul 
 10mM dNTP                  0.4ul        2.4ul    
 1mg/ml BSA                   2ul         12ul   
 Bst polymerase(8U/ul)        1ul          6ul    
 65C for 25 minutes -> keep on ice.

step8. PCR of sequencig library[edit]

                                               x6
 Nick-translated DNA          20ul             
 Solexa_PCR_up(10uM)           2ul            12ul  
 Solexa_PCR_lo(10uM)           2ul            12ul
 2xiProof master mix          50ul           300ul  
 50x SYBG                    0.8ul           4.8ul
 ddH2O                      25.2ul         151.2ul
 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.

File:20080902 sequencing library of No1 2 3 9.jpg20080902_sequencing library of No1_2_3_9

Qiaquick column purification. Yield:

 No_1: BJ, 17.4ng/ul (117nM) x 30ul
 No_2: BJ_iPS_11, 18.6ng/ul (125nM) x 30ul
 No_3: BJ_iPS_12, 32.7ng/ul (220nM) x 30ul
 No_9: Hybrid_2, 28.3ng/ul (190nM) x 30ul

Sent 15ul of each sequencing library to Yuan Gao on 08-09-02.

Blunt clone and transformation of captured PCR products and random sequencing library of No1_BJ (2008-09-04)[edit]

refer to LabNotes on [1]