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Project name
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Digestion
Reagent |
Volume |
Master Mix
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Expected: KAH126 = 1880bps MV2 = 4529bps
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15 μL/lane; 1% agarose
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Gene Ruler
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DNA(plasmid) |
3.0 μL |
-
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10X buffer |
1.5 |
4.5
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EcoRI |
1.0 |
3.0
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PstI |
1.0 |
3.0
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dH2O |
8.5 |
25.5
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- Use two 0.5mL tubes for single tubes and a 1.5ML tube for the MM
- Aliquot 12uL to each 0.5mL tube
- Add 3 uL of DNA to each
- Incubate @ 37C/>10min
How to make a gel:
- 60mL of TAE and 0.6g of agarose, swirl
- 40s in the microwave, swirl
- 40s in the microwave, swirl
- 6mL of Ethidium Bromide
- Note: Wash flask immediate
Moving onto the box
- If there is TAE buffer in the electrophoresis, put box in a casting tray
- Use B14 teeth (thinner side, thicker side is for dilute DNA). We had 2 rows.
- Check for bubbles in wells and push them out of the wells if needed
Gel Electrophoresis Procedure
- It takes 15-20 min for the gel to solidify
- At 70V is takes about 1hr for the gel to run
- Gel is done solidifying when it's translucent
- Grab comb out from both sides
- Put in runner
- Add 10uL of Ethidium Brodmide to TAE at positive end (the end facing you)
- 1KB+ loading dye (blue) > 10uL of ladder > 15uL of sample
- Pour off extra liquid and set gel on a paper towel
- Slide gel off onto UV light
- Put gel in waste box
- SIZES! Compare to expected (MV2, HPCD...) > 2 bands
Analyzing the Gel
- As a sanity check, prep 1 is more concentrated (as shown in the plate reading) and prep 2 is less bright because it is less concentrated
- Our digest was done correctly. Checking with the ladder, the bands are lining up around the expected distances. (Check the 1500 and 4000 band lines on the ladder)
- We see two bands because the plasmid was cut twice
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