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==Procedure== | ==Procedure== | ||
# | |||
# | ===Yeast Cell Lysis=== | ||
#* | #Aliquot 3uL of 0.02M NaOH into PCR tubes. | ||
# | #Using a sterile pipette tip, pick a small colony and resuspend in NaOH. | ||
## | #*If the solution is cloudy, you've added enough cells. | ||
## | #[optional] Quick-freeze the cells in liquid nitrogen | ||
#*I normally skip this step. Works just fine. | |||
#Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes. | |||
#*In the mean time, prepare the master mix for the PCR reaction. | |||
#*The boiled samples are stable at room temp for sometime. Keep on ice or freeze for longer. | |||
===PCR=== | |||
#Prepare the master mix solution containing: | |||
#*5uL 5X Q-solution | |||
#*2.5uL 10X PCR Buffer | |||
#*0.5uL dNTPs (10mM each) | |||
#*0.5uL foward primer (100uM) | |||
#*0.5uL reverse primer (100uM) | |||
#*0.25uL Taq | |||
#*12.75uL ddH2O | |||
#Aliquot 22uL of the master mix solution to each boiled sample (25uL total reaction volume). | |||
#Run the following PCR cycle: | |||
##5 min at 94C | |||
##30 cycles of: | |||
###30 sec at 94C | |||
###30 sec at 55C (or appropriate annealing temperature) | |||
###1 min/kbp at 72C | |||
##10 min at 72C | |||
==Notes== | ==Notes== |
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