PCR amplification of high GC content fragments

Summary

The following program describes in detail the PCR amplification of high GC content antique fragments. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

PCR amplification of high GC content fragments

Materials and Instruments

Betaine dNTP solution Dimethyl sulfoxide Tetramethyl sulfone Taq enzyme and enzyme buffer primers
PCR Instrument

Move

I. Materials

1. Reagents

Betaine (Sigma)

dNTP solution (four dNTPs, 25 mmol/L each)

Dimethyl sulfoxide (DMSO) (Sigma)

Tetramethyl sulfoxide (Sigma)

2. Enzyme and enzyme buffer

Taq enzyme

Taq enzyme buffer (supplied by Taq enzyme manufacturer)

3. Nucleic acids and oligonucleotides

Human genomic DNA, 20 ng per reaction.

hRF3(hGSPTl) Primer: hRF3f CCGC/CTCT/GTCG/TCGT/CGC

hRF3r CCGC/GCTG/AGGT/TCTC/CC

Klotho exon la Primer: klolaf CTCG/CAGG/TAAT/TATT/GCCAG

klolar GATG/GACG/CACC/CTTG

Klotho exon lc Primer: klolcf GTGC/AGCC/CGTG/GTCAC

klolcr GACT/CAGT/TCCC/ACAC/TTC

4. Equipment

PCR instrument

5. Other

Equipment and reagents required for agarose coagulation electrophoresis

II. Methods

1. Add the following reagents to a sterilized 0.2 ml PCR tube.

Genomic DNA 20ng

Primers (each) 15pmol

Taq enzyme 0.5U

10 x buffer 2.5ul

dNTP solution (containing four types of dNTP) 250umol/L

Add _H20 to 25ul

Contains DMSO (5%), betaine (lmol/L), betaine (2 mol/L), betaine (lmol/L) and DMS0 (5%), betaine (2 mol/L) and DMS0 (5%), and tetramethylsulfone (0.4 mol/L), depending on the treatment conditions, and the control without any other reagents.

2. Run 30 cycles using program A or B below.

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