Difference between revisions of "Haynes Lab:Notebook/Lab Traning - Pradyumna Kadambi/2013/07/02"

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(Restriction Digest)
(Restriction Digest)
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Revision as of 23:23, 7 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.