In this lab, 4 interspersed-simple-sequence-repeat (ISSR) primers were tested via performance of PCR reactions. An ISSR is a dominant marker that utilizes microsatellite repeats (followed by two arbitrary bases) as primers to amplify adjacent potions of the genome. Once amplified, the DNA pieces can be visualized on an agarose gel for length polymorphisms. Although ISSRs do not distinguish homozygotes from heterozygotes, they are useful for intraspecific population level studies.
The following ISSR markers were tested: HB10 – (GA)6CC; 17898 – (CA)6AC; Omar – (GAG)4RC; 844 – (CT)8RC. Each lab group was assigned one ISSR mentioned above. Our lab group was assigned 17898 – (CA)6AC.
5 random extracted DNA samples belonging to other students were utilized for this PCR reaction. First, 1 0.2 mL 5-tube strip (PCR tubes) was labeled with tube numbers and sample IDs (Tube 1 – PRA02; Tube 2 – PRK02; Tube 3 – GWC02; Tube 4 – CRA04; Tube 5 – PRD05). Using a p10, 1 µl of template DNA was added to the appropriate PCR tube. Pipette tips were changed between samples to prevent contamination. Once PCR tubes were filled with template DNA, they were placed on ice.
A master mix was made with the following reagents and volumes: 12.5 µl ddH2O; 3.00 µl 10x buffer +Mg; 1.00 µl BSA; 2.00 µl dNTPs; 0.25 µl 17898 primer; 0.25 µl Taq. To each amplification reaction, 19 µl of master mix was added with a p200. PCR tubes were placed into PCR machine.
Next, PCR protocol described in “Lab 10 – Plant DNA PCR I” using a primer for the psbA marker was followed due to unsuccessful results that were obtained. The following samples were used to perform the amplification reaction: PRA02; PRK02; GWC02; CRA04; PRD05. 1 0.2 mL 5-tube strip was labeled with the above sample IDs and tube number. More information regarding the protocol followed and reagents and volumes used can be found here.