Due to limited sample DNA and poor DNA quality, a semi-nested approach was used for amplification. Dye Primer and BigDye Terminator sequencing required the establishment of two different PCR protocols.
For Dye Primer sequencing, a -21M13 tailed primer was used as sense primer for the nested amplification. PCR reactions were carried out in a volume of 25µl. The used primer pair for the primary amplification was: 5’-GCA CAT AAC GGG CAG AAC GC-3’ (sense) and 5’-CCA TGA TCA CCA GGG GAA CG-3’ (antisense). The same antisense primer and 5’-TGT AAA ACG ACG GCC AGT ATG AGG CTT CCA GGC GTC CG-3’ (sense) was used for the semi-nested amplification. Use of these primers resulted in a product with a molecular size of 766 base pairs.
One international unit of Dynazyme (Dynal) was used with reaction buffers supplied by the manufacturer, 200 µM of each deoxynucleotide triphosphate (Pharmacia) and 1.5 mmol/l magnesium chloride. Primers (Table 4.3) were used at a final concentration of 0.4 µM each. Amplification was over 30 cycles at 95℃C for 30 seconds, 55℃C for 35 seconds, and 72℃C for 50 seconds. An initial denaturation step (95℃C for 2 minutes) and a final extension step (72℃C for 7 minutes) was added. One µl of this reaction was used as template for the semi-nested amplification. Semi-nested amplification was over 30 cycles at 95℃C for 30 seconds, 60℃C for 35 seconds, and 72℃C for 50 seconds. An initial denaturation step (95℃C for 5 minutes) and a final extension step (72℃C for 7 minutes) was added. Amplification was performed in 200µl tubes in a compact silver microblock of an OmniGene thermal cycler (Hybaid Ltd., England) using tube control and an oil overlay.
Five microlitres of the PCR reactions were then electrophoresed on 1.5% agarose gels and visualised with ethidium bromide staining and ultraviolet illumination.
PCR reactions were carried out in a volume of 25µl. The used primer pair for the primary amplification was: 5’-GCA CAT AAC GGG CAG AAC GC-3’ (sense) and 5’-CCA TGA TCA CCA GGG GAA CG-3’ (antisense). The same antisense primer and 5’-ATG AGG CTT CCA GGC GTC CG-3’ (sense) was used for the semi-nested amplification. Use of these primers resulted in a product with a molecular size of 748 base pairs.
One international unit of Dynazyme (Dynal) was used with reaction buffers supplied by the manufacturer, 200 µM of each deoxynucleotide triphosphate (Pharmacia) and 1.5 mmol/l magnesium chloride. Primers (Table 4.4) were used at a final concentration of 0.4 µM each. Amplification was over 30 cycles at 95℃C for 30 seconds, 50℃C for 35 seconds, and 72℃C for 50 seconds. An initial denaturation step (95℃C for 2 minutes) and a final extension step (72℃C for 7 minutes) was added. 1.5 µl of this reaction was used as a template for the semi-nested amplification. Semi-nested amplification was over 30 cycles at 95℃C for 30 seconds, 60℃C for 35 seconds, and 72℃C for 50 seconds. An initial denaturation step (95℃C for 5 minutes) and a final extension step (72℃C for 7 minutes) was added. Amplification was performed in 96 well racks in a compact silver microblock of a GeneAmp PCR System 9700 (Perkin-Elmer, England) using simulated tube control and equipped with a heated lid. This heating plate heats the air temperature at the top of each reaction mixture to a temperature that is permanently higher than the sample temperature (120℃C). This elevated air temperature relative to the sample temperature minimises evaporation so that there is no condensation of the reaction mixture as the reaction mixture is repeatedly heated and cooled. This technique eliminates the necessity of an oil overlay.
Five microlitres of the PCR reactions were then electrophoresed on 1.5% agarose gels and visualised with ethidium bromide staining and ultraviolet illumination.
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© 2001 Alexander Binder