Alice:LabNotes/2008-6-24
Experiment 1: Cosmic 3760 probe preparation
Step 1: Real time PCR for cosmic probe
Reaction System X8 X12 H2O 84.8ul 678.4 ul 1017.6 ul 10xBuffer 10ul 80 ul 120 ul SYBR (50x) 0.5ul 4 ul 6 ul *Mix the tube very well using vortex at this point to avoid locally concentrated SYBR* dNTP(10mM) 4ul 32 ul 48 ul Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul 3.2 ul 4.8 ul template(cosmic3760) 0.1ul 0.8 ul 1.2 ul Jumpstart Taq 0.2ul 1.6 ul 2.4 ul Total 100ul 800 ul 1200 ul
Primer sequence: forward primer Ap1V4IU and reverse primer Ap2V4
Reaction program:
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.
Note: 8 tubes were prepared and ran first to test if the reaction system is working properly.
Result: THe PCR reaction did not work as the curve never shot up. I am suspecting either the Taq enzyme or the 10x buffer is not working, so I will do a comparison to test the hypothesis. Since I ran a PCR reaction with Platium Taq and buffer few weeks ago, and it worked fine. So it will be used as reference.
Reaction Systems
reaction system 1: H2O 85.6ul 10xBuffer 10ul dNTP(10mM) 4ul Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul SYBR (50x) 0.5ul Platium Taq 1ul template(cosmic3760) 0.1ul Total 100ul
reaction system 2:
H2O 78ul
10x Platium Taq buffer 10ul
Jumpstart Taq polymerase 0.2ul
MgCl2(25mM) 6ul
dNTP(10mM) 4ul
primer mix (100uM) 0.4ul
50x SYBG I 0.4ul
template(cosmic 3760) 1ul
Total 100ul