Wednesday, November 19, 2003

Description
Although this method was developed for use in screening clones constructed in the pRSET family of vectors (Invitrogen), it can just as easily be used with any vector system with the correct set of primers.

Procedure
1) Pick a colony from an agar plate using a sterile 200ul pipette tip. Touch the tip to a fresh agar plate in order to duplicate the colony for later plasmid isolation. Transfer the bulk of the colony to a 1.5ml eppendorf tube containing 50ul of sterile H2O.

2) Vortex the tube to disperse the pellet and boil for 5 minutes to lyse cells and denature any DNases present.

3) Centrifuge at max. speed in a microfuge for 1 minute to remove cell debris.

4) Transfer 10ul of the supernatant to a fresh 0.5ml tube for PCR. Leave on ice until use.

5) Make the 'Master' reaction mix as follows:

Per reaction:
29ul H2O
1ul dNTP mix (10mM each of dATP, dCTP, dGTP, dTTP)
2ul primer 1 (10pmol/ul)
2ul primer 2 (10pmol/ul)
5ul 10 x PCR Buffer
1ul Taq polymerase (1U/ul)

6) Add 40ul to each sample and mix gently. Add 75ul light mineral oil to the surface of each reaction.

7) Place tubes in PCR machine and cycle using the following set up:

i) 94C for 3 minutes
ii) 55C for 30 seconds
iii) 72C for 2 minutes
iv) 94C for 15 seconds

Cycle between steps i) and iv) for 35 cycles.

8) To analyse reaction products, add 6ul 10x loading dye mix and run 10ml on an agarose gel.



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