Dinh:Protocols/Probes Prep Nov24

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Revision as of 03:29, 26 November 2009 by >Jbrubake (→‎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
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.