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
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1A: Retrieve Ice Bucket from room 240
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Retrieve 1 0.5ml tube of yeast genomic DNA and place the tube on ice
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Go Nanodrop w/ ice + Marker to find DNA concentration (ng/uL)
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label strip which says “Y-gDNA XXng/ul” + group #
Place on tube
Add to Notebook
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Place gDNA in fridge w/ PCR water
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1B: calculate the number of microliters of yeast gDNA:
100/xng/uL = uL
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place reagents on ice: PCR Master Mix, DNA Polymerase, buffer, Mg++, and all dNTPs
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Prepare 2 reaction mixes:
1. PM1-ADE2
2. PM2-18sRNA
Add MM (42uL)
Primer Mix (2.2uL)
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Label PCR tubes - Grp #.1/2/3/4
put them in the cooler with lid closed
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Set a P20 to 20ul
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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)
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Retrieve 2 sterile 0.5ml tubes - label them the same as the primer mix tubes - close tubes and vortex lightly
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Calculate the amount of water for NTC and water to add DNA to bring the total volume to 25ul
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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
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Pipet 20 ul of PM1-ADE2 reaction mix from step 3 into PCR tubes Grp#.1 and Grp#.2 and close the lids
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put info in PCR Excel Sheet
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Prepare the NTC in tubes 2 and 4 with water.
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Mix by pipetting up and down or flicking the tubes
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Prepare the reaction tubes with DNA template in tubes 1 and 3
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centrifuge to collect the volume and Bring your completed reactions in the PCR cooler to the instructor’s bench
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Lab 1
