Alice:LabNotes/2008-6-24: Difference between revisions
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Step 1: Real time PCR for cosmic probe | Step 1: Real time PCR for cosmic probe | ||
Reaction System X8 X12 | |||
H2O 84.8ul 678.4 ul 1017.6 ul | |||
H2O 84.8ul | 10xBuffer 10ul 80 ul 120 ul | ||
10xBuffer 10ul | SYBR (50x) 0.5ul 4 ul 6 ul | ||
dNTP(10mM) 4ul | *Mix the tube very well using vortex at this point to avoid locally concentrated | ||
Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul | SYBR* | ||
dNTP(10mM) 4ul 32 ul 48 ul | |||
Jumpstart Taq 0.2ul | 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 | Primer sequence: forward primer Ap1V4IU and reverse primer Ap2V4 | ||
Line 17: | Line 18: | ||
Reaction program: | 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. | 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.<br> | ||
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. | |||
<b>Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer</b> | |||
Reaction Systems | |||
reaction system 1: | |||
H2O 85.1ul | |||
10xBuffer 10ul | |||
dNTP(10mM) 4ul | |||
Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul | |||
SYBR (50x) 0.5ul | |||
Platium Taq 0.5ul | |||
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) 0.1ul | |||
Total 100ul | |||
<br> | |||
<b>Result:</b> | |||
[[Image:6-24-08 cosmic PCR.jpg]] | |||
<br> | |||
The PCR for both tubes didn't work, thus suggested that the Jumpstart Taq and enzyme were not working properly. However, a control group with platium Taq and buffer was not included, so we couldn't conclude that only the Jumpstart Taq and buffer were causing the problem. Therefore another experiment with control group will be repeated. |
Latest revision as of 17:16, 26 June 2008
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.
Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer
Reaction Systems
reaction system 1: H2O 85.1ul 10xBuffer 10ul dNTP(10mM) 4ul Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul SYBR (50x) 0.5ul Platium Taq 0.5ul 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) 0.1ul Total 100ul
Result:
File:6-24-08 cosmic PCR.jpg
The PCR for both tubes didn't work, thus suggested that the Jumpstart Taq and enzyme were not working properly. However, a control group with platium Taq and buffer was not included, so we couldn't conclude that only the Jumpstart Taq and buffer were causing the problem. Therefore another experiment with control group will be repeated.