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.