Jie:LabNotes/CpgSeq/2008-10-10

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Digestion of PGP1F with Rnase I[edit]

 PGP1F(35ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20mins.
 IMR90(25ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20min. 
 Purify with Microcon column.
 Yield: 
 IMR90 50ng/ul x 40ul. 260/280=0.96
 PGPF1 57.8ng/ul x 50ul. 260/280=0.96


Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit (4th)[edit]

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn 260:280/260:230
2 BJ_iPS_11 140ng/ul x 2 tubes 14.3ul 5.7ul 130ul 101.9ng/ul x 20ul 1.6/0.9
16 IMB90_2 50ng/ul x 2 tubes 20ul 0ul 130ul 68ng/ul x 10ul 1.48/0.93
17 PGPF1 57.8ng/ul x 3 tubes 20ul 0ul 130ul 119.5ng/ul x 30ul 1.79/1.28
  • 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;7/23/08) vol+H2O template+v2.2(24ng/ul;7/7/08) vol+H2O template+v2.3(28ng/ul;7/7/08) vol+H2O
2 BJ_iPS_11 101.9ng/ul x 20ul 1ul 4+1+1+5ul 4+1.5+4.5 8+1.5+2.5 4+1.5+3.5
16 IMB90_2 68ng/ul x 20ul 1ul 4+1+1+5ul 4+1.5+4.5 8+1.5+2.5 4+1.5+3.5
17 PGPF1 119.5ng/ul x 30ul 1ul 4+1+1+5ul 4+1.5+4.5 8+1.5+2.5 4+1.5+3.5
bis_Jurkat 200ng/ul 1ul 1+1+1+6ul
Jurkat 200ng/ul 1ul 1+ cosmic probe 2ul+h2O 6ul
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(10/13/2008)[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:20081013 captured PCR No 2 16.jpg20081013_captured_PCR_No_2_16

File:20081013 captured PCR No 17.jpg20081013_captured_PCR_No_17

Digestion of IMR_90 and PGP1F with Rnase I (10/13/08)[edit]

 PGP1F(35ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20mins.
 IMR90(25ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20min. 
 Purify with Microcon column.
 Yield: 
 IMR90 39.7ng/ul x 40ul. 260/280=1.76, 260/230=0.8
 PGPF1 72.9ng/ul x 50ul. 260/280=1.82, 260/230=1.07


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
2 BJ_iPS_11 140ng/ul x 1 tubes 14.3ul 5.7ul 130ul 147.4ng/ul x 10ul 1.6/0.9
16 IMB90_2 39.7ng/ul x 2 tubes 20ul 0ul 130ul 66ng/ul x 10ul 1.48/0.93
17 PGPF1 72.9ng/ul x 2 tubes 20ul 0ul 130ul 194.1ng/ul x 10ul 1.79/1.28
Jurkat 200ng/ul x 2 tubes 10ul 10ul 130ul 309.8ng/ul x 10ul
  • 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;7/23/08) vol+H2O template+v2.2(21ng/ul;7/23/08) vol+H2O template+v2.3(28ng/ul;7/7/08) vol+H2O
2 BJ_iPS_11 147.4ng/ul x 10ul 1ul 10+1.5
16 IMB90_2 66ng/ul x 10ul 1ul 10+1.5
17 PGPF1 191.4ng/ul x 10ul 1ul 10+1.5+2.5
bis_Jurkat 175ng/ul 1ul 1+1.5+6.5ul
bis_Jurkat 175ng/ul 1ul 1+1.5(7/7/08)+6.5
negative control 0 1ul 0+1.5+7.5

SLN mix is prepared as the above.

PCR(10/16/2008)[edit]

                                      x3
   Template                10ul      
   2X iProof Mastermix     50ul      150ul
   AmpF6.2SoL (10uM)        4ul       12ul
   AmpR6.2SoL (10uM)        4ul       12ul   
   50X SYBG I             0.8ul      2.4ul
   H2O                   31.2ul     93.6ul
   

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

File:20081016 captured PCR No 2 16 17.jpg20081016_captured_PCR_No_2_16_17


yield: No_2:

        cpg30k + suppressor: 34ng/ul x 15ul
        V2.1: 23ng/ul x 15ul
        V2.3: 13ng/ul x 15ul
        mix 2ul cpg30K + 3ul V2.1 + 5ul V2.3 = 6ng/ul x 10ul
      No_16:
        cpg30k + suppressor: 17ng/ul x 15ul
        V2.1: 13ng/ul x 15ul
        V2.3: 11ng/ul x 15ul
        mix 2.5ul cpg30k + 3ul V2.1 + 3ul V2.3 = 3ng/ul x 8.5ul
      No_17:
        cpg30k + suppressor: 16ng/ul x 15ul
        V2.1: 18ng/ul x 15ul
        V2.3: 11ng/ul x 15ul
        mix 3ul cpg30K + 3ul V2.1 + 4ul V2.3 = 5ng/ul x 10ul