Jie:LabNotes/ASE/2008-3-21

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Revision as of 17:33, 21 March 2008 by >Jie deng (New page: ==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 st...)
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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