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High Specificity PCR Amplification Using AccuPrime Taq DNA Polymerase

互联网2013-11-13

838

实验步骤

1. PCR Precautions

1) Add the following components to a sterile thin walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice:

For Small Genomic DNA ( < 200 bp), Plasmids, or cDNA):

Component	10-µl Reaction	25-µl Reaction	50-µl Reaction
10X AccuPrime^™ PCR Buffer I	1 µl	2.5 µl	5 µl
Primer Mix (10 µM each)	0.2 µl	0.5 µl	1 µl
Template DNA	10 pg–200 ng	10 pg–200 ng	10 pg–200 ng
AccuPrime^™ Taq DNA Polymerase	0.25 µl	0.5 µl	1 µl
Autoclaved distilled water	To 10 µl	To 25 µl	To 50 µl

For Genomic DNA (200 bp-4 kb):

Component	10-µl Reaction	25-µl Reaction	50-µl Reaction
10X AccuPrime^™ PCR Buffer II	1 µl	2.5 µl	5 µl
Primer Mix (10 µM each)	0.2 µl	0.5 µl	1 µl
Template DNA	1–200 ng	1–200 ng	1–200 ng
AccuPrime^™ Taq DNA Polymerase	0.25 µl	0.5 µl	1 µl
Autoclaved distilled water	To 10 µl	To 25 µl	To 50 µl

2) Mix contents of the tubes and overlay with 50 µl of mineral or silicone oil, if necessary.

3) Cap the tubes and centrifuge briefly to collect the contents.

4) Incubate tubes in a thermal cycler at 94°C for 2 min to completely denature the template and activate the enzyme.

5) Perform 25-35 cycles of PCR amplification as follows:

Denature: 94°C for 15-30 s

Anneal: 55°C-60°C for 15-30 s

Extend: 68°C for 1 min per kb<

6) Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.

7) Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.

2. Multiplex PCR Protocol

1) Add the following components to a sterile, thin-walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice:

Component	Amount
10X AccuPrime^™ PCR Buffer I (for genomic DNA <200 bp, cDNA, or plasmids) or 10X AccuPrime^™ PCR Buffer II (for genomic DNA 200 bp–4 kb)	5 µl
Primer mix (10 µM each)	1 µl each (0.2 µM each)
Template DNA	100-200 ng
AccuPrime^™ Taq DNA Polymerasundefined	1-2.5 µl
Autoclaved, distilled water	to 50 µl

2) Continue with steps 2-7 of the General Protocol.

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