Team:UNIPV-Pavia/Methods Materials/PCR

(Difference between revisions)

Revision as of 11:59, 30 June 2009

Protocols

(estimated time: 3 hours and 30 min)

For every DNA sample you want to amplify, put:
- 2 µl buffer
- 0.6 µl MgCl2
- 0.4 µl dNTPs
- 1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
- 0.2 µl Taq Polymerase
- 250 nM VF2 primer
- 250 nM VR primer
- A proper amount of ddH2O to have 20 µl of total reaction volume
into an eppendorf tube.
Put the eppendorf tube in the thermal cycler and set this program:
- 95°C 10 min
- CYCLE:
  - 95°C 30 sec
  - 60°C 1 min
  - 72°C 1-3 min
- for 35 cycles
- 72°C 7 min
- 16°C forever.
Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.

@@ Line 36: / Line 36: @@
 <br>
 <br>
-'''Materials needed:'''
+<table width='80%'><tr><td>
-*'''MgCl2'''
-*'''Buffer'''
-*'''dNTPs'''
-*'''ddH2O'''
-*'''Taq Polymerase'''
-*'''VF2 primer'''
-*'''VR primer'''
-<br>
 *For every DNA sample you want to amplify, put:
 **2 µl buffer
@@ Line 66: / Line 57: @@
 **16°C forever.
 *Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
-<br>
+<br><br><br><br>
+<br></td><td align='center' valign='bottom' width='300px'>
+<div align='center'>
+<table border='1'>
+<tr><td>
+*'''MgCl2'''
+*'''Buffer'''
+*'''dNTPs'''
+*'''ddH2O'''
+*'''Taq Polymerase'''
+*'''VF2 primer'''
+*'''VR primer'''
+</td></tr></table></div><br><br><br><br>
+</td></tr></table>