November 13

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.

Posted November 13, 2018 by Jinwoo Kim in category Uncategorized

Leave a Comment

Your email address will not be published. Required fields are marked *