StEP PCR

Evolutionary Methods in Biotechnology. Edited by S. Brakmann and A. Schwienhorst Copyright 2004 WILEY-VCH Veriag GmbH Co. KGaA, Weinlieim ISBN 3-527-30799-0 

Mix equimolar amounts of different DNA templates. Templates can be plasmids carrying the target sequence, (purified) PCR products, or DNA excised by restriction endonucleases. 

To 20 ng of total template DNA, add 5 piL of appropriate 10 x DNA polymerase buffer, 5 piL of cINTP mix, 10-100 pmol of each primer, 2.5 U Taq DNA polymerase or 1-2 U Vent DNA polymerase add CIH2O to a final volume of 50 pL. 

If there is only a single band at the correct apparent molecular weight, extract the PCR reaction mixture twice with 1 volume of phenol/chloroform/isoamyl alcohol mixture. Otherwise, see step 7. 

Perform an ethanol precipitation by adding 1/10 volume of 2.5 M sodium acetate and 2-3 volumes of 95% ice-cold ethanol. Hold the solution at —80 °C for 30 min or at —20 °C for 2 h. After centrifugation in a micro centrifuge at 4 °C and maximum speed for 30 min, carefully remove the supernatant with a mi- 

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Abecassis, V., Pompon, D. and Truan, G. (2000) High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Research, 28, E88. 

Fig. 2. Amplification of ORFs by 2-step PCR. The products of the second PCR were elec-trophoresised using a 1% agarose gel. There are some ORFs having the same 5 - and 3 - sequences, although the internal sequences are distinct (marked by ). This property is typical in wttgenes (6). These ORFs were simultaneously amplified by the same set of primers and distinguished by sequencing after cloning. Primer dimers are sometimes observed when a desired ORF is not amplified ( ). M k/Flindll marker.

Lucchi NW, Poorak M, Oberstaller J et al (2012) A new single-step PCR assay for the detection of the zoonotic malaria parasite Plasmodium knowlesi. PLoS One 7(2) e31848... 

Grandori, R., Struck, K., Giovanielli, K., and Carey, J. (1997). A three-step PCR protocol for construction of chimeric proteins. Protein Eng., 10, 1099-1100. 

The construction of chimeric enzyme genes was carried out using a self-priming PCR. High-fidelity DNA polymerase (KOD-Plus, Toyobo Biochemicals, Japan) was used in three-step PCR reactions to overcome incorporation of undesired errors during multiple PCR steps. 

Yabuki T, Motoda Y, Hanada K, et al (2007) A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis. J Struct Funct Genomics 8 173-191... 

The BC had previously been cloned from tobacco (Shorrosh et ai, 1995). We cloned the Brassica napus gene by a two step PCR protocol using high fidelity polymerase (figure 2). The 5 primer 1 was the forward sequencing primer whilst the internal primers (3 4) were derived from the available Arabidopsis EST data. The 3 primer 2 was the reverse sequencing oligonucleotide. A X ZapII rape embryo library... 

G. (2000) High efficiency family shuffling based on multi-step PCR and... 

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