Jie:LabNotes/ASE/2008-3-17
Optimization of the padlock captur efficiency[edit]
The optimal dNTP0.08nM is for SNP probe with one base gap. Now I need to verify and define the optimal dNTP concentration for SNP probe with 10 bases gap.
Design the optimization of the padlock capturing efficiency.
set the following circularization reactions: tube 1: Probe prepared on 3/12, dNTP 0.08nM tube 2: Probe prepared on 3/12, dNTP 0.8nM tube 3: Probe prepared on 3/12, dNTP 4nM tube 4: probe CES22K (370nM), dNTP 0.08nM
reaction system tube 1 tube 2 tube 3 x3 tube 4 H2O 4ul 4ul 4ul 12ul 6.7ul 10x Ampligase Buffer 1ul 1ul 1ul 3ul 1ul probe 3ul 3ul 3ul 9ul 0.3ul(CES22K,370nM) Jurkat Genomic DNA(100ng/ul) 2ul 2ul 2ul 6ul 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[edit]
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
File:2008 03 17 capture optimization.png Image:2008_03_17_capture_optimization
From left to right: Yellow is T4, Blue is T3, Green is T2, Red is T1. (Ct is 11.59 < 18.09 < 18.56 < 22.93)
From the results:
- Under the probe concentration (probe/template 47:1), the SNP probe with 10 base gap may works better in system when dNTP is 0.8nM~4nM.
- Under this condition, dNTP 0.8nM seems has no difference compared with dNTP 4nM.
- Under 0.08uM, SNP probe with 1 base gap works much better than SNP probe with 10 base gap. (I had already adjusted the two
sets of probes to get almost the same concentration for these two probe sets in the capture system)
Since the probe preparation is not so successfully this time. I will repeat the capture reaction and verify the results when I get the new probes prepared tomorrow.
Next optimization: dNTP 0.08uM, 0.8uM, 8uM, 80uM
File:2008 03 17 capture specific result.jpg
The left fragment is Kun's probe while the right one is mine. I load 4ul products of Kun's and 8ul of mine.
- The PCR products of capture with 1 base gap probe is appromately 90bp.
- The PCR product of capture with 10 base gap probe is appromately 120bp.
- 24 cycles for 1 base gap probe is too much.(too many unspecific products.)
- The optimal for 10 base gap probe is to be determined with better probe concentration.