Team:Bielefeld-Germany/Labjournal/Molecular

(Difference between revisions)

Revision as of 11:24, 6 September 2013

- Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium

- Store the colony in 4 °C while colony PCR is running

- One reaction mix contains:

- 10 µL 5x buffer

- 2 µL MgCl2 (25 mM stock)

- 1 µL dNTPs

- 0.5 µL primer mix (prefix/suffix primers or sequencing primers)

- 35.25 µL ddH2O

- 0.25 µL BioTaq polymerase (BIOLINE)

- 1 µL template

- PCR program:

- Start: 3 min, 98 °C

- 30 cycles of:

- 30 s, 98 °C

- 30 s, 55 °C

- 30 s / 1 kb template, 72 °C

- Finish: 5 min, 72 °C

Gel electrophoresis: check the fragment size

Plate the correct colony

Program Phusion
98 °C	98 °C	70 °C	72 °C	72 °C
45s	30s	1:00	45s	5:00
	35 cycles
Notes: Size: 2070bp Primer1_Gibson: Primer2_Gibson: Primer1: Primer2:

@@ Line 59: / Line 59: @@
 </div>
 <div class="standard_protocol">
+<h2>Phusion PCR</h2>
 <table border=1 style="float:left; padding-left:10px">
        <col width="150">
@@ Line 74: / Line 75: @@
 </table>
+<br><br><br><br><br><br><br><br><br><br>
+<h2>Colony PCR</h2>
+<p> - Pick one colony with a sterile tip and elute it in 100 µL ddH20 or medium </p>
+<p> - Store the colony in 4 °C while colony PCR is running <p>
+<p> - One reaction mix contains: </p>
+<p> - 10 µL 5x buffer </p>
+<p> - 2 µL MgCl2 (25 mM stock) </p>
+<p> - 1 µL dNTPs </p>
+<p> - 0.5 µL primer mix (prefix/suffix primers or sequencing primers) </p>
+<p> - 35.25 µL ddH2O </p>
+<p> - 0.25 µL  BioTaq polymerase (BIOLINE) </p>
+<p> - 1 µL template </p>
+<p> - PCR program: </p>
+<p>     - Start: 3 min, 98 °C </p>
+<p>     - 30 cycles of: </p>
+<p>        - 30 s, 98 °C </p>
+<p>        - 30 s, 55 °C </p>
+<p>        - 30 s / 1 kb template, 72 °C </p>
+<p>     - Finish: 5 min, 72 °C </p>
+<p> Gel electrophoresis: check the fragment size </p>
+<p> Plate the correct colony </p>
-<br><br><br><br><br><br><br><br><br><br><br>
+<br><br><br><br><br><br><br><br>
 </div>