Dinh:Protocols/Probes Prep Nov24: Difference between revisions

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Objectives:
Objectives:
  To determine which volume is optimal for KAPA qPCR kit reaction
  To use KAPA qPCR kit on CpG30K probes set, using reaction volume of
  To use KAPA qPCR kit on CpG30K probes set
  25ul
  To determine the efficiency of KAPA qPCR kit on lower template input
  To practice QUBIT DNA quantification
  To practice QUBIT DNA quantification
  To create a probes preparation/purification table
  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.
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:
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Revision as of 22:41, 24 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

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 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: