Jie:LabNotes/CpgSeq/2009-5-20

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Revision as of 23:32, 29 May 2009 by >Jie deng
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bisulfite conversion of PGP samples[edit]

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
09_1 NA21687*1 062507 241.1ng/ul x 1 tubes 8ul 12ul 130ul 95.4ng/ul x 10ul
09_2 NA21781*1 102307 240ng/ul x 1 tubes 8ul 12ul 130ul 120.2ng/ul x 10ul
09_3 NA21833*1 101807 253.9ng/ul x 1 tube 8ul 12ul 130ul 121.5ng/ul x 10ul
09_4 NA21660*1 053007 243.2ng/ul x 1 tube 8ul 12ul 130ul 120.3ng/ul x 10ul
09_5 NA21731*1 101807 217.4ng/ul x 1 tube 10ul 10ul 130ul 127.3ng/ul x 10ul
09_6 NA21846*1 100407 186.5ng/ul x 1 tube 11ul 9ul 130ul 151.3ng/ul x 10ul
09_7 NA21070*1 100406 329.7ng/ul x 1 tube 6ul 14ul 130ul 311.2ng/ul x 10ul
Jurkat gDNA 140ng/ul x 3 tube 14ul 6ul 130ul 255.6ng/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 reaction[edit]

sample sample concentration 10xLigase buffer template+cpg92k+H2O template+control probes+H2O reaction condition
1 bis_Jurkat 255.6ng/ul 1ul 1+4ul(#1_22.3ng/ul_05/20)+4ul 55C anneal
2 bis_Jurkat 255.6ng/ul 1ul 1+4ul(#1_22.3ng/ul_05/20)+4ul 60C anneal
3 bis_Jurkat 255.6ng/ul 1ul 1+3ul(#2_17ng/ul_05/20)+5ul 55C anneal
4 bis_Jurkat 255.6ng/ul 1ul 1+1ul(cpg30k,50ng/ul,06/13/08)+7ul 55C anneal
5 Jurkat 142.8ng/ul 1ul 1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul 55C anneal
6 09_1 95.4ng/ul 1ul 2+5ul(#2_17ng/ul_05/20)+2ul 55C anneal
7 09_7 311.2ng/ul 1ul 2+5ul(#2_17ng/ul_05/20)+2ul 55C anneal
8 negative control 0 1ul 0+4ul(#1_22.3ng/ul_05/20)+5ul 55C anneal


 dNTP concentration 10mM(NEB)
                                                                    ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP:      0.5ul         0.1ul       0.1ul       0.2ul    (10mM)0.1ul 
                                                           x30       15ul          3ul        3ul        6ul      3ul
 95c 10min -> 55C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 55C 4h -> 
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle-> 95C 5min ->
add 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold.
 tube2:  95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> 
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold.

PCR(05/24/2009)[edit]

PCR with AmpF/R6.3Sol and iProof[edit]

                                      x8
   Template                 5ul      
   2X iProof Mastermix     50ul      400ul
   AmpF6.3SoL (10uM)        4ul       32ul
   AmpR6.3SoL (10uM)        4ul       32ul   
   50X SYBG I             0.8ul      6.4ul
   H2O                   36.2ul    289.6ul
   

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 9 -> 72C 3 min -> 15C hold.

File:20090524 Jan09 captured targets iProof PCR curve.png20090524_Jan09_captured_targets_iProof_PCR_curve

File:20090524 Jan09 captured targets iProof PCR gel.jpg20090524_Jan09_captured_targets_iProof_PCR_gel

Normalization of the PCR amplicons of captured targets(2009-05-26)[edit]

  1. Purify the No09_1 and No09_7 PCR amplicons with Qiaquick column and dissolve in 30ul EB.
     concentration: 
     No09_1: 21.4ng/ul (81nM) x 30ul; 
     No09_7: 25.3ng/ul (96nM) x 30ul.
  2. Take 6ul from each sample and do the following procedure.
  3. set up the control reaction:
     12ul  water
      4ul  DSN control template
      2ul  10xDSN master buffer
     aliquot 9ul into two tubes C and E. Add 1ul DSN storage buffer into C tube. Add 1ul DSN solution into the E tube. 
Overlay with oil and incubate at 65C for 10 min. Add 5ul 2xDSN stop buffer, room temperature. 4. set up the sample reactions: 5. Add 2ul 4X Hybridization buffer (2M NaCl, 200mM HEPES), 20ul mineral oil 6. 95C 2min -> 164 cycles of (97.8C 1min, -0.2C/cycle) -> 65C 90 min. 7. Preheat 2X DSN buffer for > 5min at 65C. 8. Add 8.5ul DSN buffer to each tube, 65C 10min 9. Add 0.5ul of 1U/ul DSN enzyme to each tube, 65C 25min 10. Add 17ul 2X DSN stop buffer, 95C 10min, 4C hold. 11. Purification with Qiaquick column. The concentration of both samples are ~0.1ng/ul.

PCR with AmpF/R6.3Sol and iProof[edit]

                                      x2
   Template                 5ul      
   2X iProof Mastermix     50ul      100ul
   AmpF6.3SoL (10uM)        4ul       8ul
   AmpR6.3SoL (10uM)        4ul       8ul   
   50X SYBG I             0.8ul      1.6ul
   H2O                   40.2ul     80.4ul
   
 98C 30S -> (98C 10S -> 72C 20S) x 11 -> 72C 3 min -> 15C hold.
 After Qiaquick purification, use the nanodrop to calculate the concentration:
 No09_1: 19.3ng/ul (73nM) x 30ul;
 No09_7: 23.1ng/ul (87nM) x 30ul.

File:20090526 amplicons after normalization of captured targets.jpg20090526_amplicons after normalization of captured targets

I sent 10ul of the 09_7 NA21070*1 100406 captured targets and 09_7 NA21070*1 100406 normalized captured targets to do the end sequencing and see the read abundance of probes. The sample names are labeled as:

 21070*1 100406 captured targets: 96nM ;
 21070*1 100406 normalized : 87nM.