Jie:LabNotes/CpgSeq/2009-5-20: Difference between revisions

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   1. Purify the No09_1 and No09_7 PCR amplicons with Qiaquick column and dissolve in 30ul EB.
   1. Purify the No09_1 and No09_7 PCR amplicons with Qiaquick column and dissolve in 30ul EB.
       concentration: No09_1:         ; No09_7:
       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.
   2. Take 6ul from each sample and do the following procedure.
   3. set up the control reaction:
   3. set up the control reaction:
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       2ul  10xDSN master buffer
       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. <BR>Overlay with oil and incubate at 65C for 10 min. Add 5ul 2xDSN stop buffer, room temperature.
       aliquot 9ul into two tubes C and E. Add 1ul DSN storage buffer into C tube. Add 1ul DSN solution into the E tube. <BR>Overlay with oil and incubate at 65C for 10 min. Add 5ul 2xDSN stop buffer, room temperature.
 
   4. set up the sample reactions:
   4. set up the sample reation:
   5. Add 2ul 4X Hybridization buffer (2M NaCl, 200mM HEPES), 20ul mineral oil
   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.
   6. 95C 2min -> 164 cycles of (97.8C 1min, -0.2C/cycle) -> 65C 90 min.
Line 116: Line 117:
   9. Add 0.5ul of 1U/ul DSN enzyme to each tube, 65C 25min
   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.
   10. Add 17ul 2X DSN stop buffer, 95C 10min, 4C hold.
   11. Purification with Qiaquick column. However, the concentration is quite low ~0.1ng/ul.
   11. Purification with Qiaquick column. The concentration of both samples are ~0.1ng/ul.


===PCR with AmpF/R6.3Sol and iProof===
===PCR with AmpF/R6.3Sol and iProof===
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   98C 30S -> (98C 10S -> 72C 20S) x 11 -> 72C 3 min -> 15C hold.
   98C 30S -> (98C 10S -> 72C 20S) x 11 -> 72C 3 min -> 15C hold.
   After purification: No09_1: 19.3ng/ul;
   After Qiaquick purification, use the nanodrop to calculate the concentration:
                      No09_7: 23.1ng/ul.
 
  No09_1: 19.3ng/ul (73nM) x 30ul;
  No09_7: 23.1ng/ul (87nM) x 30ul.
 
[[Image:20090526_amplicons after normalization of captured targets.jpg]]20090526_amplicons after normalization of captured targets


===PCR with AmpF6.3NH/AmpR6.3NH and dUTP:dNTP 1:40 ===
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:
  reaction system                                                x2   
  H2O                                                38.6ul    77.2ul   
  2x Master mix                                        50ul      100ul     
  dUTP(1mM)                                            2ul        4ul     
  AmpF6.3NH(10uM)                                      2ul        4ul     
  AmpR6.3NH(10uM)                                      2ul        4ul   
  50x SYBG I                                          0.4ul      0.8ul   
  captured targets                                     5ul      5 ul each   
  Total                                              100ul      200ul   


   94C 3min -> 22 cycles of (94C 45sec -> 55C 1min -> 72C 1min) -> 72C 3min -> 4C
   21070*1 100406 captured targets: 96nM ;
  21070*1 100406 normalized : 87nM.

Latest revision as of 23:32, 29 May 2009

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.