USTC/Notebook/PCR&Colony PCR

From 2008.igem.org

(Difference between revisions)
 
(8 intermediate revisions not shown)
Line 1: Line 1:
-
<div id="header">{{Template:Team:USTC/Templates/Header}}</div>
 
-
 
'''[[Team:USTC/Notebook|< Back to Notebook]]'''
'''[[Team:USTC/Notebook|< Back to Notebook]]'''
Line 9: Line 7:
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'''  MgCl<sub>2</sub>
-
- template DNA
+
*'''0.4uL'''  dNTPs
-
(note: the total volume of primers and template can be 0.5 to 20µL)
+
*'''200nM'''  final concentration of each primer VF2 and VR (0.2uL)
-
 
+
*'''0.2uL'''  Taq enzyme
 +
*''template DNA''
 +
(note: add ddH<sub>2</sub>O to 20µL, the total volume of PCR is 20µL)
3. make sure reaction tubes are properly capped before placing in thermocycler
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
-
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
+
== 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
 +
*'''0.4 uL''' MgCl<sub>2</sub>
 +
*'''2 uL''' PCR Buffer  
 +
*'''2 uL''' colony culture  
 +
*'''0.2 uL''' Taq enzyme
 +
*'''15.8 uL''' H<sub>2</sub>0
-
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

Latest revision as of 16:11, 27 October 2008

< Back to Notebook

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 VF2 and VR (0.2uL)
  • 0.2uL Taq enzyme
  • template DNA

(note: add ddH2O to 20µL, 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
  • 0.4 uL MgCl2
  • 2 uL PCR Buffer
  • 2 uL colony culture
  • 0.2 uL Taq enzyme
  • 15.8 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

Retrieved from "http://2008.igem.org/USTC/Notebook/PCR%26Colony_PCR"