930
edits
Changes
Created page with '==Reagents== *Primers *dNTPs (2 mM of each dNTP) *Expand polymerase (Roche) *Primers *Yeast Colonies ==Protocol== #Pick about 20% of a colony and mix in 20 uL of sterile water. ...'
==Reagents==
*Primers
*dNTPs (2 mM of each dNTP)
*Expand polymerase (Roche)
*Primers
*Yeast Colonies
==Protocol==
#Pick about 20% of a colony and mix in 20 uL of sterile water.
#Prepare PCR:
*5 uL Expand Polymerase Buffer
*5 uL dNTPs
*1 uL of a 5X diluted stock
*1 uL of a 5X diluted stock
*5 uL cell suspension
*0.5 uL Expand Polymerase
===Run PCR Program Colony PCR:===
#94C for 5 min
#94C for 10s
#40C for 30s
#68C for 2 min
#Repeat Steps 2-4 10 times
#94C for 10s
#40C for 30s
#68C for 1 min + 20s/cycle
#Repeat Steps 6-8 19 times
#68C for 7min
#4C hold forever
*Check PCR by Agarose Gel Electrophoresis
*Primers
*dNTPs (2 mM of each dNTP)
*Expand polymerase (Roche)
*Primers
*Yeast Colonies
==Protocol==
#Pick about 20% of a colony and mix in 20 uL of sterile water.
#Prepare PCR:
*5 uL Expand Polymerase Buffer
*5 uL dNTPs
*1 uL of a 5X diluted stock
*1 uL of a 5X diluted stock
*5 uL cell suspension
*0.5 uL Expand Polymerase
===Run PCR Program Colony PCR:===
#94C for 5 min
#94C for 10s
#40C for 30s
#68C for 2 min
#Repeat Steps 2-4 10 times
#94C for 10s
#40C for 30s
#68C for 1 min + 20s/cycle
#Repeat Steps 6-8 19 times
#68C for 7min
#4C hold forever
*Check PCR by Agarose Gel Electrophoresis
