Team:Macquarie Australia/Protocols/GibsonAssembly

From 2013.igem.org

(Difference between revisions)
Line 9: Line 9:
<html>
<html>
-
Before beginning Gibson assembly, all agar plate requirements, concentration calculations and equipment requirements were prepared. Once the gBlocks had arrived Gibson assembly was performed on all fragments.<br><br>
 
 +
Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidlines.
 +
<br><br>
-
Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. The table below shows each tube with vector & volume, volume of Gibson Master Mix, water volume & volume of each gBlock fragment. After incubation, these were transformed into Top10 cells using the Transformation Protocol found here - 
+
NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:
-
</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
+
<br><br>
<br><br>
-
<br/>
+
0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
-
'''5. After addition of all components (shown in table) incubation was preformed at 50°C for 60 min followed. '''
+
<br><br>
-
<br/>
+
 
 +
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:
 +
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
 +
<br><br>
 +
 
 +
The cells were appropriately transformed and concentrations were determined prior. (transformation protocol can be found with the following link)
 +
-</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
 +
<br><br>
 +
<html>
 +
<center><h1><td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
 +
.</h7></center>
 +
<br><br>
 +
 
 +
After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min. '''
 +
 
<br/>
<br/>

Revision as of 02:54, 27 September 2013


Gibson Assembly Protocol


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

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

0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.

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)

The cells were appropriately transformed and concentrations were determined prior. (transformation protocol can be found with the following link) -Transformation Protocol.



.



After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min. '''
Element ChlMChlI1CTH1PORChlGDVR1ChlPChlI2GeneGene
Fragment 13.3uL Gblock15.0uL Gblock12.3uL Gblock14.8uL Gblock1
Fragment 23.3uL Gblock23.3uL Gblock23.2uL Gblock23.0uL Gblock2
Fragment 33.1uL Gblock3
10X T4 DNA ligase buffer2 µL
Vector (0.05pmol)2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL2.7uL
H2O0.7uL
Gibson Master Mix10 uL11 uL11.3 uL10.5 uL

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