User:Hana Benzeer/Notebook/SGM Summer Project/2011/07/18

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Digestion of purified I15009

To 30μL plasmid:

  1. Add 0.5μL of BSA
  2. Add 5μL buffer 3
  3. Add 1μL of XbaI
  4. Add 1μL of PstI
  5. Add 12.5μL Free Dnase water
  6. Leave it in incubator for 2hrs

Prepare 1% Agarose gel

  1. weigh 0.65g of Agarose powder into 200ml conical flask
  2. Add 65ml of EDTA buffer.
  3. Swirl to mix
  4. Heat for 2 min in microwave to dissolve the agarose. Note= until no specals are observed
  5. Leave it to cool on the bench for 5 minutes down to about 60°C
  6. Add 2μL of Ethidium Bromide and swirl to mix. Note= Ethidium Bromide is mutagenic and should be handled with extreme caution. Dispose of the contaminated tip into a dedicated ethidium bromide waste container.
  7. Pour the gel slowly into the tank. Note the Comb is already positioned int its place. Push any bubbles away to the side using a #disposable tip. Note=The benefit of pouring slowly is that most bubbles stay up in the flask
  8. Leave to set for at least 1 hour

Preparing the samples

To 50μL digested I15009 X/P Note= the total volume is 50μL because purified sample is 30μL in whch the materials are added in total of 20μL.

  1. Add 10μL of 6x loading buffer giving a total of 60μL into I15009 X/P.
  2. Mix well.
  3. Add 10μL of 1kb ladder into first well
  4. Leave the second well blank
  5. Add 55μL of the sample into the third well
  6. Leave the fourth well blank
  7. Add 5μL of the rest sample into the fifth well
  8. Close the gel tank, switch on the power-source and run the gel at 100 volt and 60 min
  9. Check that a current is flowing (ie. bubbles).

PCR of I15009 and I15010

2x each so 4x in total. 4 PCR reactions

    1. 2x I15009
    2. 2x I15010