Team:Macquarie Australia/Protocols/GibsonAssembly

From 2013.igem.org

(Difference between revisions)
(Created page with "{{Team:Macquarie_Australia/Style2}} {{Team:Macquarie_Australia/Header}} <center> <h1>Gibson Assembly Protocol</h1> </center>")
 
(15 intermediate revisions not shown)
Line 4: Line 4:
<center>
<center>
<h1>Gibson Assembly Protocol</h1>
<h1>Gibson Assembly Protocol</h1>
 +
</center>
</center>
 +
 +
 +
<html>
 +
<center> <h7>
 +
Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on <b>NEB guidelines</b>. </center>
 +
<br><br>
 +
 +
<b>NOTE:</b> For efficiency with reference to the NEB Gibson protocol, we considered:
 +
<br><br>
 +
 +
<font size=3><b>1)</b></font> 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
 +
<br>
 +
 +
<font size=3><b>2)</b></font>  0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.
 +
<br><br>
 +
 +
A calculation provided was used furthermore to determine the pmols required based on the length of fragments: <br>
 +
<b>pmols</b> = (weight in ng) x 1,000 / (base pairs x 650 daltons)
 +
<br><br>
 +
 +
 +
<table border="0" cellpadding="10">
 +
<tr>
 +
 +
<td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
 +
<td>
 +
 +
 +
<center><font size=3><b>Legend</b></font></center>
 +
 +
<br>
 +
<font size=5>*</font> =      50ng of 5000 bp dsDNA is approx 0.015 pmols.
 +
<br><br>
 +
<font size=5>**</font> =  Control reagents
 +
<br><br>
 +
<font size=5>***</font> =  Additional master mix may be required for larger bp fragments.
 +
<br><br>
 +
<b> Note - </b>  50ng of 500 bp is approx 0.15 pmol
 +
<br><br>
 +
<b> Note - </b>  50-100ng of vector recommended with excess insert of 2-3 fold
 +
<br><br>
 +
<b> Note - </b>  Use 5 x more insert if  <200bps.
 +
<br><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
 +
<br>
 +
<b>After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.</b>

Latest revision as of 10:15, 27 September 2013


Gibson Assembly Protocol


Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidelines.


NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:

1) 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
2) 0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.

A calculation provided was used furthermore to determine the pmols required based on the length of fragments:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)

Legend

* = 50ng of 5000 bp dsDNA is approx 0.015 pmols.

** = Control reagents

*** = Additional master mix may be required for larger bp fragments.

Note - 50ng of 500 bp is approx 0.15 pmol

Note - 50-100ng of vector recommended with excess insert of 2-3 fold

Note - Use 5 x more insert if <200bps.


After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.

Retrieved from "http://2013.igem.org/Team:Macquarie_Australia/Protocols/GibsonAssembly"