Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi/2013/07/02

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(Electrophoresis)
Current revision (02:10, 8 July 2013) (view source)
(Electrophoresis)
 
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#Clean off UV light.
#Clean off UV light.
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==Notes==
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*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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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
  1. Thaw fast digest buffer, and take out X Enzyme and P Enzyme from the fridge.
  2. 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)
  3. Label a 0.5mL centrifuge tube with initials and plasmid number. (PK KAH013)
  4. 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.
  5. Make sure to add the largest volumes first.
  6. 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.
  7. Centrifuge the solution using the minicentrifuge.
  8. Place the centrifuge tube in a dry bath(hot plate) at 37 degrees for 10 mins.


Electrophoresis

Perparation of Gel

  1. 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)
  2. Add 60mL of 1xTAE buffer, and mix thoroughly with agarose.
  3. Microwave flask for 30s.
  4. 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.
  5. Microwave for 30s again, and repeat the swirling.
  6. Wait for agarose gel liquid to cool.

Preparation of Gel Plates

  1. Make sure you are wearing gloves and a lab coat, you will be handling ethidium bromide!
  2. Take ethidium bromide out of fridge, and with a paper towel open the ethidium bromide.
  3. Add 6μL ethidium bromide to the agar liquid in flask, and swirl carefully.
  4. Set up mold by ensuring that the mold is securely assembled and is leak proof.
  5. Place comb in mold.
  6. Pour agar liquid gel in the flask into mold.
  7. Wait for gel to set.
  8. Remove gel from mold carefully without removing the comb. Take care not to tear the gel.
  9. Transfer gel to electrophoresis apparatus.
  10. Fill both sides of the electrophoresis apparatus with 1xTAE to the indicated line.
  11. Then remove comb.

Perform Electrophoresis

  1. Using a micropipette, add 10μL of Ladder into the left-most well. Make sure no bubbles form in the pipette tip.
  2. When adding DNA to wells, angle the micropipette, and try make sure that the DNA does not leave the well.
  3. 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)
  1. Make sure that the black and red pins are correctly connected to the apparatus, black towards the wells, and red towards the other side.
  2. Set apparatus to 100V, and press run.
  3. Wait 1 hour.
  4. Gently pull gel out of the apparatus taking care not to tear it, and dry it and place it in paper towels.
  5. Place gel under UV light, and close the lid on the UV light. Record needed observations, and remove the gel.
  6. 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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