Team:UNIPV-Pavia/Methods Materials/PCR

(Difference between revisions)

Revision as of 16:38, 22 September 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.

MgCl2

Buffer

dNTPs

ddH2O

Taq Polymerase

VF2 primer

VR primer

@@ Line 56: / Line 56: @@
 <table class='in_pcr' border='1'>
 <tr><td>
-*'''MgCl2'''
+<font class='label'>
-*'''Buffer'''
+'''MgCl2'''<hr color='white' width='70%'>
-*'''dNTPs'''
+'''Buffer'''<hr color='white' width='70%'>
-*'''ddH2O'''
+'''dNTPs'''<hr color='white' width='70%'>
-*'''Taq Polymerase'''
+'''ddH2O'''<hr color='white' width='70%'>
-*'''VF2 primer'''
+'''Taq Polymerase'''<hr color='white' width='70%'>
-*'''VR primer'''
+'''VF2 primer'''<hr color='white' width='70%'>
+'''VR primer'''</font>
 </td></tr></table><br><br><br><br>
 </td></tr></table>