Jie:LabNotes/ASE/2008-3-21
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Optimization of the padlock captur efficiency
I will do for 10 base gap again. considering the suggestion from Billy, I will try the dNTP 0.08nM and 0.8nM for 1 base gap probe (one step annealing); to 1.5nM for 20 cycyles annealing. Try the dNTP 0.08nM, 0.8nM, 4nM and 40nM for 10 base gap probe.
Design the optimization of the padlock capturing efficiency.
set the following circularization reactions: tube 1: Probe prepared on 3/19, dNTP 0.08nM tube 2: Probe prepared on 3/19, dNTP 0.8nM tube 3: Probe prepared on 3/19, dNTP 4nM tube 4: probe prepared on 3/19, dNTP 40nM tube 5: probe CES22K (370nM), dNTP 0.08nM tube 6: probe CES22K (370nM), dNTP 0.8nM
reaction system tube 1 tube 2 tube 3 tube 4 x4 tube 5 tube 6 H2O 4ul 4ul 4ul 4ul 12ul ul 10x Ampligase Buffer 1ul 1ul 1ul 1ul 4ul 1ul probe 3ul 3ul 3ul 3ul 0.3ul (CES22K,370nM) Jurkat Genomic DNA(100ng/ul) 2ul 2ul 2ul 2ul 8ul 2ul
Reaction program:
Tube 1: 95c 10min -> 60C 1h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP) -> 60C 1h
-> (95c 1min -> 60C 1h) x 20cycles -> 37C 1min -> add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. Tube 2: same as tube1 accept that 0.8nM dNTP Tube 3: same as tube1 accept that 4pM dNTP tube 4: same as tube1
prepartion of SLN 1ul: Storage reagents: AmpliTaq Stoffel fragment(10U/ul), AmpLigase(5U/ul),dNTP(0.5uM),dNTP(10nM),dNTP(0.2nM)
*add 1ul dNTP(0.5uM) to 249ul ddH2O to get 2nM dNTP ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.08nM dNTP 0.2ul 0.1ul 0.1ul 0.2ul (0.2nM)0.4ul 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.8nM dNTP: 0.2ul 0.1ul 0.1ul 0.2ul (2nM)0.4ul 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 4nM dNTP: 0.2ul 0.1ul 0.1ul 0.2ul (10nM)0.4ul prepare 10x(ddH2O 6ul, Ligase Buffer 3ul, Ligase 3ul, Amplitaq 6ul, mix and aliquot into 3 tubes,
then add dNTP 4ul with 0.2nM, 2nM and 10nM dNTP respectively.
PCR
PCR reaction system
reaction system x8 H2O 30.6ul 244.8ul 10x JumpStartTaq Buffer 5ul 40ul MgCl2(25mM) 3ul 24ul dNTP(10mM) 2ul 16ul AmpSolV6F2/R2 mix(10uM)(no tail) 2ul 16ul 50x SYBG I 0.4ul 3.2ul JumpStartTaq 2ul 16ul Products of Circluarization 5ul 40ul Total 50ul 400ul
Run the program on Opticon4 realtime thermocycler 94C 3min -> 24 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 4C hold