PCR for knockouts and epitope tags

Chris Tachibana April 2004, modified August 2004

• references:

Güldener, U et al (1996) NAR 24:2519 for pUG6

www.uni-frankfurt.de/fb15/mikro/euroscarf/data/Del_plas.html for pAG series

Knop M et al (1999) Yeast 15: 963 for epitope tagging

• For knockouts use plasmids pUG6 for a kanmx knockout or the pAG series for other markers. For epitope tagging use the pYM series.

• Use primers at 0.3µM final concentration. Make a stock that is 3µM.

• Use the Roche Expand Long Template PCR systerm (1 681 842) in the -20 enzyme freezer, small white plastic boxes. Keep the DNA polymerase mix cold.

• Not included in the mix is dNTPs. Our standard stock is 2mM and the final concentration will be 0.35mM. Keep them cold.

• Use0.5µl plasmid DNA per reaction.

• Make at least 4 x 50µl reactions. Recipe for 4 reactions:

20µl 10X buffer 1

100 µl sterile H₂O

35 µl 2mM dNTPs

20 µl forward primer

20 µl reverse primer

2µl plasmid DNA

3µl DNA polymerase mix

distribute into 4 x 50µl PCR tubes and put in the PCR machine

• Use the Robocycler, linked programs 31-36 OR the MJ Research DNA engine program LONG-PCR. Takes 4-5 hours to run.

IMPORTANT for the Robocycler:

1. Look at the bottom of the screen and make sure temp mode is 1, gradient is OFF and link pgm is ON. If you need to change these settings, hit shift + extended functions. Go to 1 to change the temp mode to “start at set temperature” and 3 to change the linked programs.

2. If you have to stop the machine (for example if it takes off and is running before you are ready and you have to stop it with shift + exit to put your samples in), you have to RESET THE LINKED PROGRAMS AND RESTART THE HOT TOP.

3. At least 10 minutes before starting, hit the red button on the hot top so it starts warming up. Before starting the run, make sure the red and green lights are on.

• Check 1-2µl on a 0.8% agarose gel before transforming.