November 7, 2018

Preparation of Lupinus arboreus samples for PCR amplification using interspersed simple sequence repeat (ISSR) primers was performed during this phase of the project. Using ISSRs, the ultimate objective is to determine whether populations can be grouped more closely through geographical proximity or flower color. The following procedure was used to prepare 1:10 dilutions of the five Lupinus arboreus samples provided:

1. Five samples of Lupinus arboreus samples were given (PRA01 to PRA05).
2. Fifteen 1.5-mL tubes were obtained and labeled with the ID code, three new tubes for each original sample.
3. 45-μL of H2O was placed into each newly labeled tube.
4. 5-μL of each sample was pipetted into three of the correspondingly labeled dilution tubes, switch pipette tips across samples.
5. One of each dilution tube was placed into a separate rack for dispersal.

After completing the dilution process, the preparation of the template DNA and Master Mix (MM) was performed:

1. Two 0.2-mL 8-tube PCR strips were obtained for the fifteen reactions.
2. Completed the PCR sheet by indicating the tube number and corresponding sample ID.
3. Tubes were labeled on the cap with information matching that of the PCR sheet, including additional details such as primer number and initials written along the sides of the tubes
4. 1-μL of diluted DNA from each sample was pipetted into the corresponding PCR tubes, following the order of the PCR sheet. Pipette tips were changed between samples.
5. Once completed, the lid was gently closed atop the tubes and the strips were placed on ice.
6. To create the master mix, reagent amounts were calculated for 70 reactions, resulting in a MM of 875μL of ddH2O, 210μL of 10x buffer + Mg, 70μL of BSA, 140μL of dNTPs, 17.5μL of primer OMAR, and 17.5μL of Taq (provided by the Professor). Reagents were added to a 1.5-mL tube labeled MM for Master Mix in the listed order.
7. 19-μL of the MM were pipetted into each PCR tube, pipetting up and down to mix the MM with the diluted sample. Tips were changed for each sample, and the last tube (tube 16) contained only the MM to act as a negative control.
8. PCR tube strips were capped tightly then placed on a rack for PCR.