Polymerase Chain Reaction for the Identification of a Plasmid

Add 1ng of the target DNA,

5µL of each primer,

25µL of 2X PCR master mix,

and sufficient sterile water to make the volume up to 50µL.

Thermocycle on the following settings:

A	B	C
1 cycle	94	1
30 cycles	94	1
30 cycles	55	1
30 cycles	72	1
1 cycle	72	5

cycle programme for PCR

Weigh out the correct amount of agarose and place it in a 250mL conical flask. Note: for a 1% gel you will need 1g of agarose for every 100mL of buffer.

Add 100mL 1× TAE buffer and swirl gently to mix. Put an inverted small conical flask in the top of the flask.

Place in a microwave and heat for 1 minute, then gently swirl the contents.

Continue microwaving in 30-second blasts followed by gentle swirling until the agarose starts to boil.

Continue microwaving in 15-second blasts until all the agarose has dissolved - you should not be able to see any tiny pieces floating in the gel suspension.

Place the flask in a 50°C water bath to cool. It is important to make sure the agarose has cooled to 50°C before proceeding to the next stage.

Whilst you are waiting for the gel to cool, prepare your gel tray by securely taping up the ends.

Once the agarose has cooled (5-10 minutes should be OK), add 4µL of to the cooled gel mixture and swirl to mix.

Pour the molten agarose into the gel tray and insert the comb (make sure it is straight and level) and leave to set.

Add TAE buffer to the tank, then add the gel and ensure it is fully submerged in TAE.

Load the DNA samples into the gel, and add a DNA ladder to one of the wells

Run the gel at 100V for 1h 0m 0s

Observe the gel and locations of the DNA fragments