Alice:LabNotes/2008-6-30

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Experiment 1: Cosmic probe preparation using Thermopol buffer[edit]

Real time PCR reaction system:
                                                                x12
H2O                                                81.1ul      973.2 ul
10X Thermopol buffer                                 10ul        120 ul
50x SYBG I                                          0.4ul        4.8 ul
dNTP(2.5mM)                                           8ul         96 ul       
primer mix (100uM)                                  0.2ul        2.4 ul
template(cosmic 3760)                               0.1ul        1.2 ul
Platium Taq polymerase                              0.2ul        2.4 ul
Total                                               100ul       1200 ul
PCR program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> go to step 2 x 21 cycles -> 72C 5min -> 15C hold.

Result:
File:6-30-08 cosmic w thermopol buffer.jpg
The PCR reaction started to have some activity during very early stage and shoot up around 15th cycle. However, the fluorescence level is not high enough. Therefore I suspect this buffer might not be as efficient as original platium buffer, since the pre-activity might cause some unspecific bands. Therefore I tried with another iTaq buffer on next expriment to see if the result is better.

Experiment 2: Cosmic probe preparation using iTaq buffer[edit]

Real time PCR reaction system:
                                                                x12
H2O                                                74.9ul      973.2 ul
10X iTaq buffer                                      10ul        120 ul
MgCl2(25mM)                                           6ul         72 ul
50x SYBG I                                          0.4ul        4.8 ul
dNTP(2.5mM)                                           8ul         96 ul       
primer mix (100uM)                                  0.2ul        2.4 ul
template(cosmic 3760)                               0.1ul        1.2 ul
Platium Taq polymerase                              0.2ul        2.4 ul
Total                                               100ul       1200 ul
PCR program: 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> go to step 2 x 21 cycles -> 72C 5min -> 15C hold.

Result:
File:6-30-08 cosmic w iTaq.jpg
The reaction shoot up at 15th cycle, and the curve seems to be better than thermopol buffer. Even though the fluorescence level is not much greater than thermopol buffer, but the overall result seems to be better.