Dinh:Protocols/Probes Prep Nov24
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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 |
2. 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
3. Stopped at X cycles.