Digestion of purified I15009
To 30μL plasmid:
- Add 0.5μL of BSA
- Add 5μL buffer 3
- Add 1μL of XbaI
- Add 1μL of PstI
- Add 12.5μL Free Dnase water
- Leave it in incubator for 2hrs
Prepare 1% Agarose gel
- weigh 0.65g of Agarose powder into 200ml conical flask
- Add 65ml of EDTA buffer.
- Swirl to mix
- Heat for 2 min in microwave to dissolve the agarose. Note= until no specals are observed
- Leave it to cool on the bench for 5 minutes down to about 60°C
- 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.
- 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
- 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.
- Add 10μL of 6x loading buffer giving a total of 60μL into I15009 X/P.
- Mix well.
- Add 10μL of 1kb ladder into first well
- Leave the second well blank
- Add 55μL of the sample into the third well
- Leave the fourth well blank
- Add 5μL of the rest sample into the fifth well
- Close the gel tank, switch on the power-source and run the gel at 100 volt and 60 min
- Check that a current is flowing (ie. bubbles).
PCR of I15009 and I15010
2x each so 4x in total. 4 PCR reactions
- 2x I15009
- 2x I15010