Dinh:Protocols/Probes Prep Nov24

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Revision as of 19:26, 25 November 2009 by >Alan6017518 (→‎=Step 1: PCR Amplification)
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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

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
novel enzyme in kit enhances the amplification efficiency of both high
 GC and AT templates
Chromo4 RT Detector does not require ROX Reference dye

Objectives:

To use KAPA qPCR kit on CpG30K probes set, using reaction volume of
 25ul
To determine the efficiency of KAPA qPCR kit on lower template input
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.

Step 1: PCR Amplification

Reaction Mix (0.33pg input):

Reagent Final Conc. Vol (1x) Vol (8x)
ddH20 11.806ul 94.5ul
2x KAPA qPCR Master Mix 1x 12ul 96ul
AP1V4IU (100uM) 0.3uM 0.072ul 0.58ul
AP2V4 (100uM) 0.3uM 0.072ul 0.58ul
Template CpG30k (20uM) 0.33pg 0.05ul 0.4ul
Total 24ul 192ul

2. Program

94C - 30 s
94C - 3 s
60C - 30 s
Plate Read
Cycle to 2 for 40 more times
72C - 5 min
15C - forever
end

3. Stopped at X cycles.