Alice:LabNotes/2008-5-26: Difference between revisions

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(New page: Experiment 1: Run a agarose gel on cosmic 3760 probe Image:ZhangLab_2 2008-05-26 14hr 45min.jpeg Result: the gel had consistent result with the PCR curves, which no product was produc...)
 
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Experiment 1: Run a agarose gel on cosmic 3760 probe
Experiment 1: Run a agarose gel on cosmic 3760 probe<br>
 
[[Image:ZhangLab_2 2008-05-26 14hr 45min.jpeg]]
[[Image:ZhangLab_2 2008-05-26 14hr 45min.jpeg]]


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Experiment 2: prepare a cosmic probe with Taq master mix   
Experiment 2: prepare a cosmic probe with Taq master mix   
Experiment 1: Padlock Library preparation for probe 27K
Experiment 1: Padlock Library preparation for probe 27K
Experiment 1: Padlock Library preparation for probe 27K
Experiment 1: Padlock Library preparation for probe 27K


Step 1: Real time PCR
Step 1: Real time PCR


   reaction system                                  
   reaction system                                 x2 
   H2O                              48.9ul                  
   H2O                              48.9ul       97.8       
   Taq master mix(2x)                50ul          
   Taq master mix(2x)                50ul       100 
   forward + reverse primer(100uM)  0.5ul                        
   forward + reverse primer(100uM)  0.5ul       1             
   SYBR Green (50x)                  0.5ul            
   SYBR Green (50x)                  0.5ul       1
   probe template (cosmic 3760)      0.1ul          
   probe template (cosmic 3760)      0.1ul       0.2
   Total                            100ul        
   Total                            100ul       200
   
   
   
   

Latest revision as of 16:11, 20 June 2008

Experiment 1: Run a agarose gel on cosmic 3760 probe

File:ZhangLab 2 2008-05-26 14hr 45min.jpeg

Result: the gel had consistent result with the PCR curves, which no product was produced from the amplification. However, the gel should have some primer dimers, but none is shown. This is possibly caused by not loading enough samples. The first lane is the ladder, which had 0.5 ul of 100 bp ladder, diluted with 3.5 ul H2O, and 1 ul of 5% TBE dye. The rest of the lanes had 4 ul of PCR product, and 1 ul of 5% TBE dye.

Experiment 2: prepare a cosmic probe with Taq master mix

Step 1: Real time PCR

 reaction system                                 x2   
 H2O                              48.9ul        97.8         
 Taq master mix(2x)                 50ul        100   
 forward + reverse primer(100uM)   0.5ul        1              
 SYBR Green (50x)                  0.5ul        1 
 probe template (cosmic 3760)      0.1ul        0.2
 Total                             100ul        200


Reaction program:

94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold.