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

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(Restriction Digest)
(Electrophoresis)
Line 36: Line 36:
==Electrophoresis==
==Electrophoresis==
 +
<u>Perparation of Gel</u><br>
 +
#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.
 +
<u>Preparation of Gel Plates</u><br>
 +
#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.
 +
#Using a micropipette, add 10
 +
<u></u><br>
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Revision as of 01:43, 8 July 2013

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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.
  12. Using a micropipette, add 10



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