Blackburn:Yeast Colony PCR

(Difference between revisions)

Revision as of 00:19, 12 June 2006

This is a quick and easy yeast colony PCR protocol that does not require zymolyase step.

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.

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:
1. 5 min at 94C
2. 30 cycles of:
  1. 30 sec at 94C
  2. 30 sec at 55C (or appropriate annealing temperature)
  3. 1 min/kbp at 72C
3. 10 min at 72C

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@@ Line 11: / Line 11: @@
 ==Procedure==
-#Step 1
-#Step 2
+===Yeast Cell Lysis===
-#*Step 2 has some additional information that goes with it.  i.e. Keep at 4&deg;C.
+#Aliquot 3uL of 0.02M NaOH into PCR tubes.
-#Step 3
+#Using a sterile pipette tip, pick a small colony and resuspend in NaOH.
-##Step 3 has multiple sub-steps within it.
+#*If the solution is cloudy, you've added enough cells.
-##Enumerate each of those.
+#[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==