Lab 11 Entry
11/7/18 Wednesday Plant DNA PCR-ISSRs
Professor Paul ran our Genomic DNA of the samples from last class, several samples were successful and some weren’t.
In this lab, we used two interspersed-simple-sequence-repeat (ISSR) primers in PCR reaction. We used samples of Lupinus arboreus samples from last years class. It was the same species that we saw in our field trips.
First, I made 1:10 dilutions of 5 Lupinus arboreus samples. Three 1.5 mL tubes with unique ID. I pipette 45 micro liters of water and 5 micro liters of each samples into each of the three 1.5 mL tubes. Then I put total of 15 tubes into a rack so that every table gets the diluted samples.
After everyone was done with dilution, I grabbed 15 diluted tubes: PRL 01,PRL 02, PRL 03, PRL 04, PRL 05, PRM 01, PRM 02, PRM 03, PRM 04, PRM 05, PHT 01, PHT 02, PHT 03, PHT 04, and PHT 05. I then grabbed two 0.2 ml 8-tube strips (PCR tube strips) that I will be using for the 15 reactions per marker. I wrote sample ID, primer number (17898) and my initials. I then using filter tips, I pipette 1 micro liter of my first template DNA (dilution) into the first tube of my PCR strips that I labeled. I continued this step and changed the tips everytime I changed my samples. After I was done transferring 1 micro liters each into 15 PCR strips, I put them on ice.
Kayla V. then created Master Mix for our table. Our Master Mix was based on per 80 reactions so the measurements were ddh2o=1000 micro liters, 10x buffer +mg=240 micro liters, BSA=80 micro liters, dNTPs=160 micro liters, Primer (17898)=20 micro liters, Taq=20 micro liters and my template=1 micro liter. Total of 1520 except the amount of my template DNA.
After she was done creating the Master Mix, I pipette 19 micro liters of the Master Mix and added to each of the PCR strips adding up to 20 micro liters into each PCR strips. Every time I added the Master Mix, I mixed the solution by gently pipetting up and down. After pipetting in 19 micro liters of the Master Mix into each PCR strips (15 time), I closed the lid tightly and put them into the PCR machine.
That was the end of the lab on Wednesday.