Jie:LabNotes/ASE/2008-3-17

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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.