Jie:LabNotes/ASE/2008-2-29: Difference between revisions

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PCR reaction system
PCR reaction system


   reaction system                                    
   reaction system                                                       x14
   H2O                                                36.2ul                                  36.2ul
   H2O                                                36.2ul                                  36.2ul
   10x JumpStartTaq Buffer                            4.5ul                                  4.5ul
   10x JumpStartTaq Buffer                            4.5ul                                  4.5ul
  MgCl2()
   dNTP(10mM)                                            1ul                                    1ul
   dNTP(10mM)                                            1ul                                    1ul
   AmpSolV6F2/R2 mix(10uM)                              2ul                                    2ul
   AmpSolV6F2/R2 mix(10uM)                              2ul                                    2ul
   50x SYBG I                                          0.2ul                                  0.2ul
   50x SYBG I                                          0.2ul                                  0.2ul
   JumpStartTaq                                          1ul                                    1ul   
   JumpStartTaq                                          1ul                                    1ul   
   Products of Circluarization                                                 5ul                                    5ul
   Products of Circluarization                           5ul                                    5ul


Run the program on Opticon4 realtime thermocycler
Run the program on Opticon4 realtime thermocycler

Revision as of 22:00, 27 February 2008

during the circularization reaction, the ratio of probe/target should be at least 125:1 or 250:1.
for example: genomic DNA is 100ng/ul. How much CES22K probe(360, concentration is 9.3ng/ul) is needed?

 since the molecular wight of one genome is 3pg, 200ng DNA is equal to 200ng/3pg.
the probe molecule needed is 200ng/3pg x 250. For each target and the total probe molecule is 200ng/3pg x 250 x 22,000.
the Mol of the probe is 200ng/3pg x 250 x 22,000 / (6x10 23) the concentration of probe is 9.3ng/ul. The length of each probe is approximately 90base (single strand oligoDNA). Since 1base = 330Da, the concentration of probe is 9.3ng/ul /(90x330) = 300nM the volume the probe needed is 400ng/3pg x 250 x 22,000 / {(6x10 23 阿佛加德罗常数)x70nM} = 2.0ul

Now add 1.5ul probe in reaction, the ratio of probe/DNA = 1.5x250/2 =188


 set the following circularization reactions:
 tube Standard 1: Standard reaction system
 tube Standard 2: Standard reaction system, Alu I incubation before PCR
 tube Standard 3: Standard reaction system, control for Alu I incubation before PCR 
 tube 1: decrease dNTP concentration from  to 50uM to 1uM 
 tube 2: decrease dNTP concentration from  to 50uM to 0.02uM
 tube 3: change the annealing steps from 60C overnight to the mutiple steps
 tube 4: increase probe concentration from ratio 188:1 to 752:1
   
 reaction system                Stad tube 1   Stad tube 1   Stad tube 3    tube 1    tube 2     tube 3     tube 4
 H2O                              5.5ul         5.5ul          5.5ul       5.5ul      5.5ul     5.5ul        1ul 
 10x Ampligase Buffer               1ul           1ul            1ul         1ul        1ul       1ul        1ul
 probe                            1.5ul         1.5ul          1.5ul       1.5ul      1.5ul     1.5ul        6ul
 Jurkat Genomic DNA(100ng/ul)       2ul           2ul            2ul         2ul        2ul       2ul        2ul

Reaction program:

 Standard 1: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h -> 37C 1min 
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 Standard 2: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h -> 37C 1min 
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> add 0.5ul RE-AluI (100nM), the ALuI guide oligo -> denaturing at 94C 3min -> 37C 1h -> 75C 10min -> 4C hold.
 Standard 3: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h -> 37C 1min 
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> add 0.5ul ddH2O -> denaturing at 94C 3min -> 37C 1h -> 75C 10min -> 4C hold.
 tube 1: same as standard except that dNTP in SLN mix is 25uM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP) 
 tube 2: same as standard except that dNTP in SLN mix is 10uM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.008nM dNTP) 
 tube 3: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h -> (95c 1min -> 60C 1h x 20cycles)
-> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 tube 4: 95c 10min -> 60C overnight -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2uM dNTP) -> 60C 1h -> 37C 1min 
-> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
 prepartion of SLN is needed calculated.
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1uM dNTP
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.02uM dNTP

PCR

PCR reaction system

 reaction system                                                        x14
 H2O                                                36.2ul                                  36.2ul
 10x JumpStartTaq Buffer                             4.5ul                                  4.5ul
 MgCl2()
 dNTP(10mM)                                            1ul                                    1ul
 AmpSolV6F2/R2 mix(10uM)                               2ul                                    2ul
 50x SYBG I                                          0.2ul                                  0.2ul
 JumpStartTaq                                          1ul                                    1ul  
 Products of Circluarization                           5ul                                    5ul

Run the program on Opticon4 realtime thermocycler 94C 3min -> 8 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 14 cycles of (94C 30sec -> 72C 1min) -> 72C 3min -> 4C hold

waiting for Alu 2 oligo