Dinh:Protocols/Probes Prep Nov24: Difference between revisions
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3. Stopped at X cycles. | 3. Stopped at X cycles. | ||
==Notes:== | |||
8x reaction mix was prepared as test run. 100ul was aliquoted to 6 tubes and amplified using RT-PCR for 26 cycles (over-amplified), amplification slope started at cycle 13 and plateaued at cycle 25. For the rest of the reaction mix, 25ul was aliquoted to 8 tubes. Only 4 tubes were amplified using the the same program for 26 cycles (over-amplified), amplification sloped started also at cycle 13 and plateaued at cycle 25. Concluded no difference between 25ul and 100ul reaction volumes. | |||
The leftover 4 tubes of 25ul reaction volume was used in another run: | |||
Program | |||
95C - 30 s | |||
95C - 3 s | |||
60C - 30 s | |||
72C - 3 s | |||
Plate Read | |||
Cycle to 2 for 29 more times | |||
72C - 5 min | |||
15C - forever | |||
end | |||
Recommended by KAPA qPCR manual: 95C denaturation (initital & in cycle), and 72C during plate read. | |||
However, for this run, amplification slope started at cycle 15 and plateaued also at cycle 23. Concluded that other program used was better suited for this probe set. |
Revision as of 03:43, 26 November 2009
November 24, 2009
KAPA SYBR FAST qPCR Kit Master Mix (2x) Universal
ideally suited for gene expression analysis low copy gene detection microarray validation gene knockdown validation novel enzyme in kit enhances the amplification efficiency of both high GC and AT templates
Notes:
protect kit components from light always ensure that the product has been fully thawed and mixed before use do not exceed 25 ul reaction volumes do not exceed 20ng template DNA per 24 ul reaction volume Chromo4 RT Detector does not require ROX Reference dye
Objectives:
To use KAPA qPCR kit on CpG30K probes set, using reaction volume of 25ul and 100ul To practice QUBIT DNA quantification To create a probes preparation/purification table
Previous methods to quantify probes concentration at each purification steps included Nanodrop and PAGE quantification. However, Nanodrop quantification is inaccurate after ethanol precipitation. The drawback to PAGE quantification is that it cannot determine the total DNA concentration. I will use QUBIT and PAGE to determine the efficiency of each probes preparation step.
November 25, 2009
Step 1: PCR Amplification
Reaction Mix (100ul reaction volume):
Reagent | Final Conc. | Vol (1x) | Vol (8x) | Vol (100x) |
---|---|---|---|---|
ddH20 | 49.4ul | 395ul | 4940ul | |
2x KAPA qPCR Master Mix | 1x | 50ul | 400ul | 5000ul |
AP1V4IU (100uM) | 0.3uM | 0.3ul | 2.4ul | 30ul |
AP2V4 (100uM) | 0.3uM | 0.3ul | 2.4ul | 30ul |
Template CpG30k (20nM) | 33pg | 0.05ul | 0.4ul | 5ul |
Total | 100.05ul | 800.4ul | 10005ul |
Program
94C - 30 s 94C - 3 s 60C - 30 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end
Protocol:
Place KAPA 2x qPCR MM on ice. Add ddH20, primers, and template to 15ml tube (RNAase/DNAase free). Vortex & leave on ice for 2-3 min Prepare strip tubes on cooling rack Add KAPA 2x MM & vortex, shake down reagents, then pour into 55ml solution basin Use multichannel p-100 pipettor. Pipette 101ul to each tube. For last row, use p-200 to pipette 101ul to each tube. Place caps on strip tubes Spin pairs of strip tubes for 5-10 s to remove bubbles Prepare new run on Chromo4 RT-PCR machine, let it get to 94 C Pause program, place tubes in machine, and resume run
3. Stopped at X cycles.
Notes:
8x reaction mix was prepared as test run. 100ul was aliquoted to 6 tubes and amplified using RT-PCR for 26 cycles (over-amplified), amplification slope started at cycle 13 and plateaued at cycle 25. For the rest of the reaction mix, 25ul was aliquoted to 8 tubes. Only 4 tubes were amplified using the the same program for 26 cycles (over-amplified), amplification sloped started also at cycle 13 and plateaued at cycle 25. Concluded no difference between 25ul and 100ul reaction volumes.
The leftover 4 tubes of 25ul reaction volume was used in another run:
Program
95C - 30 s 95C - 3 s 60C - 30 s 72C - 3 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end
Recommended by KAPA qPCR manual: 95C denaturation (initital & in cycle), and 72C during plate read.
However, for this run, amplification slope started at cycle 15 and plateaued also at cycle 23. Concluded that other program used was better suited for this probe set.