Restriction Digest
| Reagent |
Volume
|
| DNA- KAH013 Plasmid |
10.0 μL
|
| dH2O |
6.0
|
| 10x Fast Digest Buffer |
2.0
|
| X Enzyme |
1.0
|
| P Enzyme |
1.0
|
| |
20 μL
|
- Thaw fast digest buffer, and take out X Enzyme and P Enzyme from the fridge.
- Place X Enzyme and P Enzyme in blue block to keep them cold. ( Block turns yellow/green if it is warmed, make sure block does not warm)
- Label a 0.5mL centrifuge tube with initials and plasmid number. (PK KAH013)
- Using a micropipette, Add 10μL of DNA(KAH013 plasmid), 6μL of dH2O , 2μL 10x Fast Digest buffer, 1μL X Enzyme, and 1μL P Enzyme to the centrifuge tube.
- Make sure to add the largest volumes first.
- When adding in the X and P Enzymes, take care not to remove the enzymes from the cooling block. Angle the micropipette and the tube of enzyme such that the tube enzyme does not leave the cooling block.
- Centrifuge the solution using the minicentrifuge.
- Place the centrifuge tube in a dry bath(hot plate) at 37 degrees for 10 mins.
Electrophoresis
Perparation of Gel
- Add 0.6g of agarose to the flask specifically labeled for preparation of agarose gel. (Add agarose first so that it does not stick to the sides of the flask)
- Add 60mL of 1xTAE buffer, and mix thoroughly with agarose.
- Microwave flask for 30s.
- Remove the flask from the microwave using hot hands, point it away from you and swirl. Make sure to swirl away the buildup on the sides of the flask.
- Microwave for 30s again, and repeat the swirling.
- Wait for agarose gel liquid to cool.
Preparation of Gel Plates
- Make sure you are wearing gloves and a lab coat, you will be handling ethidium bromide!
- Take ethidium bromide out of fridge, and with a paper towel open the ethidium bromide.
- Add 6μL ethidium bromide to the agar liquid in flask, and swirl carefully.
- Set up mold by ensuring that the mold is securely assembled and is leak proof.
- Place comb in mold.
- Pour agar liquid gel in the flask into mold.
- Wait for gel to set.
- Remove gel from mold carefully without removing the comb. Take care not to tear the gel.
- Transfer gel to electrophoresis apparatus.
- Fill both sides of the electrophoresis apparatus with 1xTAE to the indicated line.
- Then remove comb.
Perform Electrophoresis
- Using a micropipette, add 10μL of Ladder into the left-most well. Make sure no bubbles form in the pipette tip.
- When adding DNA to wells, angle the micropipette, and try make sure that the DNA does not leave the well.
- Add 10μL of KAH013 DNA into a different well.
- Order follwed of DNA placed in wells was: Ladder, David's plasmid (KAH06), Prad's plasmid (KAH013), and Heather's Plasmid(KAH014)
- Make sure that the black and red pins are correctly connected to the apparatus, black towards the wells, and red towards the other side.
- Set apparatus to 100V, and press run.
- Wait 1 hour.
- Gently pull gel out of the apparatus taking care not to tear it, and dry it and place it in paper towels.
- Place gel under UV light, and close the lid on the UV light. Record needed observations, and remove the gel.
- Clean off UV light.
Notes
- Electrophoresis appears to be successful because my DNA and David's DNA traveled almost the same distance. The plasmid I used and the plasmid David used were less that 50 base pairs different in size.
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Project name
