USTC/Notebook/PCR&Colony PCR

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(Difference between revisions)
Line 9: Line 9:
1. set up pre-labeled reaction tubes on ice
1. set up pre-labeled reaction tubes on ice
-
 
2. add the following components:
2. add the following components:
-
- 45µL Platinum PCR SuperMix (rock gently after thawing, quick spin before use)
+
- 2µL  PCR buffer (rock gently after thawing, quick spin before use)
-
- 200nM final concentration of each primer (insert calculation short cut here)
+
- 1.2uL  Mgcl2
 +
- 0.4uL  dNTPs
 +
- 200nM final concentration of each primer  
 +
- 0.2uL Taq enzyme
- template DNA
- template DNA
-
(note: the total volume of primers and template can be 0.5 to 20µL)
+
(note: the total volume of PCR is 20µL)
Line 21: Line 23:
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4. perform PCR with an initial heating step at 94C for 2 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C
+
4. perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C
== colony PCR ==
== colony PCR ==
-
2 uL primer #1 (to 10uM) 2 uL primer #2 0.4 uL DNTPs 2 uL Buffer 5 uL colony culture 0.2 uL Taq 10.4 uL H20  
+
0.2 uL primer #1 (to 25uM)  
 +
0.2 uL primer #2
 +
0.4 uL dNTPs
 +
2 uL Buffer  
 +
5 uL colony culture  
 +
0.2 uL Taq  
 +
10.4 uL H20  
-
92C 1:30 (92 0:30 50 :30 72 1:30) repeat x30 72 10:00
+
perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min

Revision as of 13:43, 27 October 2008

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PCR

Briefly, a typical reaction is set up as follows:


1. set up pre-labeled reaction tubes on ice

2. add the following components: - 2µL PCR buffer (rock gently after thawing, quick spin before use) - 1.2uL Mgcl2 - 0.4uL dNTPs - 200nM final concentration of each primer - 0.2uL Taq enzyme - template DNA (note: the total volume of PCR is 20µL)


3. make sure reaction tubes are properly capped before placing in thermocycler


4. perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C


colony PCR

0.2 uL primer #1 (to 25uM) 0.2 uL primer #2 

0.4 uL dNTPs

2 uL Buffer 5 uL colony culture 0.2 uL Taq 10.4 uL H20

perform PCR with an initial heating step at 94C for 5 minutes followed by 25-35 cycles of 30sec at 94C, 30sec at 55C and 1Kb/min at 72C,and 72C for 10min

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