Master GREEN 200 Manual de usuario Pagina 24

  • Descarga
  • Añadir a mis manuales
  • Imprimir
  • Pagina
    / 56
  • Tabla de contenidos
  • MARCADORES
  • Valorado. / 5. Basado en revisión del cliente
Vista de pagina 23
2-4 PCR
Amplifying Custom Target Sequences for Quantitation
Overview
We recommend the following steps for the development of real-time
quantitative PCR assays:
Ordering Reagents
See Materials Required but Not Supplied on page 1-3 for a list of
required reagents and equipment.
Quantitating
Primers
Use a spectrophotometric method to determine the concentrations of
the primers received:
Measure the absorbance at 260 nm of a 1:100 dilution of each
oligonucleotide in TE buffer.
Calculate the oligonucleotide concentration (C) in µM using the
method shown in the table on the next page.
Step Action See Page
1 Order Reagents 2-4
2 Quantitate Primers 2-4
3 Optimize Primer Concentrations for:
PCR
One-Step RT-PCR
Two-Step RT-PCR
4-2
4-5
4-9
Chromophore
Extinction
Coefficient Number
Extinction
Coefficient
Contribution
A 15,200 1 15,200
C 7050 6 42,300
G 12,010 5 60,050
T 8400 6 50,400
Total
—— 167,950
Absorbance (260 nm) = sum of extinction coefficient contributions × cuvette
pathlength × oligonucleotide concentration/100
0.13 = 167,950 M
-1
cm
-1
× 0.3 cm × C/100
C=258µM
Vista de pagina 23
1 2 ... 19 20 21 22 23 24 25 26 27 28 29 ... 55 56

Comentarios a estos manuales

Sin comentarios