Endy:Yeast Colony PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Barry Canton (talk | contribs) No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
''Back to [[Yeast Colony PCR]]'' | ''Back to [[Yeast Colony PCR]]'' | ||
==Method== | ==Method== | ||
#Using sterile pipette tips, transfer a 1 mm colony into 50 uL of 60 U/ml | #Using sterile pipette tips, transfer a 1 mm colony into 50 uL of 60 U/ml Zymolyase | ||
#*3 uL of 1 U/mL | #*3 uL of 1 U/mL Zymolyase stock solution | ||
#*47 uL of water | #*47 uL of water | ||
#Incubate cell suspension at 37 C for 30 minutes. | #Incubate cell suspension at 37 C for 30 minutes. | ||
| Line 9: | Line 9: | ||
'''Zymolase Stock Solution''' | '''Zymolase Stock Solution''' | ||
*10,000 U | *10,000 U Zymolyase (Sigma) | ||
*1 mL KHPO4 buffer, pH 7.5 (adjusted with KOH) | *1 mL KHPO4 buffer, pH 7.5 (adjusted with KOH) | ||
*0.2 mL 5M NaCl | *0.2 mL 5M NaCl | ||
Revision as of 13:03, 16 February 2006
Back to Yeast Colony PCR
Method
- Using sterile pipette tips, transfer a 1 mm colony into 50 uL of 60 U/ml Zymolyase
- 3 uL of 1 U/mL Zymolyase stock solution
- 47 uL of water
- Incubate cell suspension at 37 C for 30 minutes.
- Incubate cell suspension at 95 C for 10 minutes.
- Use 5 uL of the cell suspension for a 50 uL PCR reaction
Zymolase Stock Solution
- 10,000 U Zymolyase (Sigma)
- 1 mL KHPO4 buffer, pH 7.5 (adjusted with KOH)
- 0.2 mL 5M NaCl
- 5 mL 100% glycerol
- Add H2O to 10 mL
- Store at -20 C
