Jie:LabNotes/ASE/2008-3-21: Difference between revisions

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>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...)
 
>Jie deng
(Removing all content from page)
 
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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<BR> -> (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,<BR>  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

Latest revision as of 21:10, 24 March 2008