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

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==PCR(05/24/2009)==
==PCR(05/24/2009)==
 
===PCR with AmpF/R6.3Sol and iProof===
                                       x8
                                       x8
     Template                5ul       
     Template                5ul       
     2X iProof Mastermix    50ul      400ul
     2X iProof Mastermix    50ul      400ul
     AmpF6.2SoL (10uM)        4ul      32ul
     AmpF6.3SoL (10uM)        4ul      32ul
     AmpR6.2SoL (10uM)        4ul      32ul   
     AmpR6.3SoL (10uM)        4ul      32ul   
     50X SYBG I            0.8ul      6.4ul
     50X SYBG I            0.8ul      6.4ul
     H2O                  36.2ul    289.6ul
     H2O                  36.2ul    289.6ul
Line 94: Line 94:
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.


===2nd PCR amplification with AmpF6.3NH/AmpR6.3NH and dUTP:dNTP 1:40 ===
===PCR with AmpF6.3NH/AmpR6.3NH and dUTP:dNTP 1:40 ===
   
   
   reaction system                                                x2     
   reaction system                                                x2     
Line 106: Line 106:
   Total                                              100ul      200ul     
   Total                                              100ul      200ul     


   94C 3min -> 22 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
   94C 3min -> 22 cycles of (94C 45sec -> 55C 1min -> 72C 1min) -> 72C 3min -> 4C

Revision as of 17:43, 24 May 2009

bisulfite conversion of PGP samples

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

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 16h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 55C 4h -> 
4 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 1h -> 94C 5min -> 4C hold.
 tube2:  95c 10min -> 60C 16h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> 
4 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 1h -> 94C 5min -> 4C hold.

PCR(05/24/2009)

PCR with AmpF/R6.3Sol and iProof

                                      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 10-> 72C 5 min -> 15C hold.

PCR with AmpF6.3NH/AmpR6.3NH and dUTP:dNTP 1:40

 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