Endy:Yeast Colony PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Barry Canton (talk | contribs) No edit summary |
No edit summary |
||
| (2 intermediate revisions by 2 users not shown) | |||
| Line 1: | Line 1: | ||
#Using sterile pipette tips, transfer a 1 mm colony into 50 uL of 60 U/ml | ''Back to [[Yeast Colony PCR]]'' | ||
#*3 uL of 1 U/mL | ==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 | #*47 uL of water | ||
#Incubate cell suspension at 37 C for 30 minutes. | #Incubate cell suspension at 37 C for 30 minutes. | ||
| Line 7: | 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 | ||
| Line 13: | Line 15: | ||
*Add H2O to 10 mL | *Add H2O to 10 mL | ||
*Store at -20 C | *Store at -20 C | ||
==Related Topics== | |||
[[Silver: Colony PCR]] | |||
[[Category:Yeast]] [[Category:Protocol]] | |||
Latest revision as of 06:02, 10 September 2009
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
