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
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 20 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 determine which volume is optimal for KAPA qPCR kit reaction To use KAPA qPCR kit on CpG30K probes set 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.