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4-2 Optimizing Primer Concentrations

Optimizing Primer Concentrations for PCR

Overview

The purpose of the procedure below is to determine the minimum

primer concentrations giving the lowest threshold cycle (C

) and

maximum

∆R

while minimizing nonspecific amplification. The reaction

volumes are 50 µL. Use 10 to 100 ng of genomic DNA or 1 to 10 ng of

cDNA template.

PCR Master Mix is used in the procedure on page 4-2 to run four

replicates of each of the nine conditions shown in the table below. The

master mix is described in “PCR Master Mix for Primer Optimization” on

page 4-3.

Optimizing Primer

Concentrations for

PCR

Reverse

Primer (nM)

Forward Primer (nM)

50 300 900

50 50/50 300/50 900/50

300 50/300 300/300 900/300

900 50/900 300/900 900/900

To optimize primer concentrations for PCR:

Step Action

1 Load the plate for both a template and a No Template Control

(NTC) matrix, as shown in “Plate Configuration for Primer

Optimization for PCR” on page 4-3.

2 Place the plate in the appropriate ABI P

RISM

Sequence Detection

System.

Use the thermal cycling conditions in “Thermal Cycling Parameters

for Primer Optimization” on page 4-4.

Note SYBR Green must be calibrated on the instrument. Please

refer to the appropriate instrument User’s Manual to calibrate the

instrument with SYBR Green.

3 At the end of the run:

Tabulate the results for the yield. This analysis will identify the

optimum concentrations of primers for PCR yield.

Tabulate the results for the C

value. This analysis will identify

the optimum primer concentrations for C

and for the absence of

nonspecific amplification.

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