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Science & NatureLab 1
By derrickousley
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Expanded Abstract Lab #2 - What: Performing PCR, In Silico, and Agarose Gel Analysis Why: Verifying there is pure yeast DNA in high yield How: 2 ways - Absorption Spec @ 250nm and Agarose Gel Electrophoresis Methods: PCR w/ 2 primer pairs (PM1- target for CRISPR) then In Silico PCR (computer PCR with both primer pairs to see expected fragment size) Results: Agarose should show fragment size and verify DNA is yeast Materials: ice bucket, PCR Water, Pipets, 0.5ml tubes, Pipet Tips, Microfuge tube rack, sharpie, PCR mix = MM, Ade2for/Aderev mix = PM1, NS1/NS2 mix = PM2, PCR cooler, 4tube PCR strip, and yeast gDNA

1A: Retrieve Ice Bucket from room 240
Retrieve 1 0.5ml tube of yeast genomic DNA and place the tube on ice
Go Nanodrop w/ ice + Marker to find DNA concentration (ng/uL)
label strip which says “Y-gDNA XXng/ul” + group # Place on tube Add to Notebook

Place gDNA in fridge w/ PCR water

1B: calculate the number of microliters of yeast gDNA: 100/xng/uL = uL
place reagents on ice: PCR Master Mix, DNA Polymerase, buffer, Mg++, and all dNTPs
Prepare 2 reaction mixes: 1. PM1-ADE2 2. PM2-18sRNA Add MM (42uL) Primer Mix (2.2uL)
Label PCR tubes - Grp #.1/2/3/4 put them in the cooler with lid closed
Set a P20 to 20ul

Additional items: • One tube of 10uM Ade2for/Ade2rev primer mix (PM1) • One tube of 10uM NS1/NS2 primer mix (PM2) • One PCR Cooler rack (small blue freezer tray) • One PCR tube strip (4 tubes)
Retrieve 2 sterile 0.5ml tubes - label them the same as the primer mix tubes - close tubes and vortex lightly
Calculate the amount of water for NTC and water to add DNA to bring the total volume to 25ul
Change Pipet tip - Pipet 20 ul of PM2-18sRNA reaction mix from step 3 into PCR tubes Grp#.3 and Grp#.4 and close the lids
Pipet 20 ul of PM1-ADE2 reaction mix from step 3 into PCR tubes Grp#.1 and Grp#.2 and close the lids

put info in PCR Excel Sheet
Prepare the NTC in tubes 2 and 4 with water.
Mix by pipetting up and down or flicking the tubes

Prepare the reaction tubes with DNA template in tubes 1 and 3

centrifuge to collect the volume and Bring your completed reactions in the PCR cooler to the instructor’s bench