Verifying Kan Deletion Mutants from Genomic DNA

Materials

• Order primers to amplify the knockout from the genome. The primers should be ~21 bases long and about 200 to 300 bp upstream from the start and another one that is 200-300 bp from the stop
• Order KanC primer (Forward primer starting at 1003 bp of KANR region.) 5’-> 3’ TGA TTT TGA TGA CGA GCG TAA T
• Order KanB primer (Reverse primer ending at 344 bp of KANR region.) 5’->3’ CTG CAG CGA GGA GCC GTA AT
• when you receive all the primers resuspend them to 100 pmol/ul in ddH2O

The idea is to check with KanC forward primer and your genomic downstream reverse primer. Then also check with Kan B reverse primer and your genomic upstream primer.

Grow up yeast cultures to isolate the Genomic DNA from the Yeast. See Rapid Isolation of Genomic DNA from Yeast. Use 1µl of the DNA in your PCR reaction.

PCR Reaction

• 1 uL Genomic DNA
• 2.5 uL Expand Long Template Buffer 1
• 1.25 uL of a 1:10 dilution of the forward primer
• 1.25 uL of a 1:10 dilution of the reverse primer
• 0.5 uL dNTP mix (10mM stock)
• 1 uL Expand Long Template Enzyme
• 17.5 uL ddH2O
• 25 µl Total Volume

PCR Program

• Make sure your annealing temperature is 5C lower than the lowest Tm of your two primers

1. 94C 10 sec
2. annealing temp 30 sec
3. 68C 1 min
4. Go to 1, 9 times
5. 94C 10 sec
6. annealing temp 30 sec
7. 68C 1 min + 20 sec/cycle
8. Go to 5, 19 times
9. 68C 7 min
10. 4C forever

Check for the correct size band on an agarose gel.