ISSR amplification reactions made last week using 5 random extracted DNA samples (belonging to other students) with the 17898 ISSR were used for gel electrophoresis. 1 µl of loading dye was added to each PCR reaction tube using a p10. Pipette tips were changed between each sample.
Using a pipette of the same size, 10 µl of each DNA-loading-dye mixture was added to the appropriate well. The gel was run at 60volts for 1.5 hours.
After the run, the gel tray was scanned to determine successful ISSRs.
Successful ISSRs included Omar and 17898.
Next, 1:10 dilutions using our extracted DNA samples (Lab 9) were made. 5 1.5mL centrifuge tubes were labeled with the corresponding sample ID and “1:10.” Labels are as follows: PRK01 1:10; PRK02 1:10; PRK03 1:10; PRK04 1:10; PRK05 1:10. Using a p10, 10 µl of sample DNA was added to the appropriate centrifuge tube. A p200 was used to add 90 µl of ddH2O to each tube. Assembling dilutions ensured the concentration of potentially present plant secondary compounds were minute, as these compounds could affect PCR success.
Two 0.2 mL 5-tube strips for 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 strip 1 were used to perform PCR using 17898 and Omar was assigned to strip 2.
1 µl of 1:10 diluted DNA was added to the corresponding tubes of strips 1 and 2 using a p10. Pipette tips were changed after each sample.
For each ISSR, 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: 12.5 µl ddH2O; 3.00 µl 10x buffer +Mg; 1.00 µl BSA; 2.00 µl dNTPs; 0.25 µl primer; 0.25 µl Taq. The primer corresponding to each ISSR was added to the appropriate master mix. Volumes were multiplied by 20 to compensate for the quantity of reactions for the group.
A p200 was used to transfer 19 µl of the master mix containing the 17898 ISSR to each tube of strip 1. 19 µl of the master mix containing the Omar ISSR was added to each tube of strip 2. Finally, strips were placed into PCR machine.