Verifying Kan Deletion Mutants from Genomic DNA - Revision history

Davebrid: Added categories

2012-02-21T16:39:53Z

Added categories

← Older revision		Revision as of 16:39, 21 February 2012
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			[[Category:Genotyping]]
			[[Category:Yeast]]
			[[Category:PCR]]
			[[Category:DNA]]

	==Materials==		==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 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

Davebrid: changed PCR reaction to a list

2012-02-21T16:39:06Z

changed PCR reaction to a list

← Older revision		Revision as of 16:39, 21 February 2012
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	==PCR Reaction==		==PCR Reaction==
	1 uL Genomic DNA		* 1 uL Genomic DNA
	2.5 uL Expand Long Template Buffer 1		* 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 forward primer
	1.25 uL of a 1:10 dilution of the reverse primer		* 1.25 uL of a 1:10 dilution of the reverse primer
	0.5 uL dNTP mix (10mM stock)		* 0.5 uL dNTP mix (10mM stock)
	1 uL Expand Long Template Enzyme		* 1 uL Expand Long Template Enzyme
	17.5 uL ddH2O		* 17.5 uL ddH2O
	25 µl Total Volume		* 25 µl Total Volume

	==PCR Program==		==PCR Program==

Davebrid: copied protocol from emily kauffman

2012-02-21T16:38:33Z

copied protocol from emily kauffman

New page

==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.