Jie:LabNotes/ASE/2008-2-29

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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 0.2uM to 4nM 
 tube 2: decrease dNTP concentration from  to 0.2uM to 0.08nM
 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 (CES22K,#390)              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 1 except that dNTP in SLN mix is 4nM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP) 
 tube 2: same as standard 1 except that dNTP in SLN mix is 0.08nM (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; 0.2uM dNTP
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM 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