Lab 10 – Plant DNA PCR I

The first step in this lab was to load gels using DNA extracted from plant samples previous lab. A p10 was used to pipette 5 “dots”of loading dye, each of 1 µl onto Parafilm. Using a pipette of the same size, 3 µl of the template DNA was added to the appropriate loading dye “dot.” Once the extracted DNA was added, a p10 set at 4.1 µl was used to transfer the template and loading dye into the wells to perform a gel electrophoresis.

The gels were run at 130 volts for about 25 minutes.  After the run, the gel tray was scanned.

Next, DNA extracted from the samples of Lupinus aarboreus in the previous lab was used to perform PCR. Two different PCR reactions were assembled for each sample. One PCR reaction used a primer for the Internal-Transcribed Spacer (ITS) marker found in Fungi. The second reaction used a primer specific to the chloroplast gene, psbA.

Two 0.2 mL 5-tube strips suitable for PCR were labeled with the appropriate sample ID (tubes of strip 1 were labeled as follows: PRK01, PRK02, PRK003, PRK04, PRK05 and strip 2 was labeled as PRK11, PRK12, PRK13, PRK14, PRK14, PRK15). Contents in tube 1 were used to perform PCR for the ITS marker and the psbA marker was assigned to strip 2. Using a new filter pipette for each sample, 1 µl of template DNA was added to the corresponding tube of both strips. Once the DNA was added to all tubes, the strips were placed on ice.

For each marker, a master mix was made that included all the reagents necessary to perform the PCR reaction. By creating a master mix, the likelihood of errors associated with pipetting small volumes were minimized. The ingredients and associated volumes used for each PCR reaction are as follows: 15.0 µl ddH2O; 2.00 µl 10x buffer + Mg; 1.00 µl BSA; 0.20 µl dNTPs; 0.20 µl forward primer, 0.20 µl reverse primer; 0.04 µl Taq. The forward and reverse primers corresponding to each marker were added to the corresponding master mix. Volumes were multiplied by 20 to compensate for the table’s reactions and negative control reactions.

19 µl of the ITS master mix was added to all tubes of strip 1. Likewise, 19 µl of the psbA master mix was added to each tube of strip 2. Finally, PCR tubes were closed and placed into the PCR machine.

 

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