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 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; 50uM 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; 50uM 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; 50uM 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; 1uM dNTP) tube 2: same as standard except that dNTP in SLN mix is 10uM (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.02uM dNTP) tube 3: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM 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; 50uM 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 H2O 36.2ul 36.2ul 10x JumpStartTaq Buffer 4.5ul 4.5ul 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