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Changes
copied protocol from emily kauffman
==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
# 94C 10 sec
# annealing temp 30 sec
# 68C 1 min
# Go to 1, 9 times
# 94C 10 sec
# annealing temp 30 sec
# 68C 1 min + 20 sec/cycle
# Go to 5, 19 times
# 68C 7 min
# 4C forever
Check for the correct size band on an agarose gel.
*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
# 94C 10 sec
# annealing temp 30 sec
# 68C 1 min
# Go to 1, 9 times
# 94C 10 sec
# annealing temp 30 sec
# 68C 1 min + 20 sec/cycle
# Go to 5, 19 times
# 68C 7 min
# 4C forever
Check for the correct size band on an agarose gel.
