Template:SBB-Protocols GGAssembly

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(New page: ===Golden Gate Assembly=== Reaction mix: <pre> 0.5uL of each plasmid* 1uL T4 DNA ligase buffer 0.5uL T4 DNA ligase 0.5uL BsaI 5.5uL water 10uL total volume </pre> Ideal is equimolar amoun...)
Current revision (15:47, 29 January 2014) (view source)
(Golden Gate Assembly)
 
Line 2: Line 2:
Reaction mix:
Reaction mix:
<pre>
<pre>
-
0.5uL of each plasmid*
+
2uL of each plasmid*
1uL T4 DNA ligase buffer
1uL T4 DNA ligase buffer
0.5uL T4 DNA ligase
0.5uL T4 DNA ligase
0.5uL BsaI
0.5uL BsaI
-
5.5uL water
+
6uL water
10uL total volume
10uL total volume
</pre>
</pre>

Current revision

Golden Gate Assembly

Reaction mix:

2uL of each plasmid*
1uL T4 DNA ligase buffer
0.5uL T4 DNA ligase
0.5uL BsaI
6uL water
10uL total volume

Ideal is equimolar amounts of each plasmid, but if your mini preps are consistent, you don’t really have to bother normalizing.

Thermocycler program:

37C for 2min
16C for 5min
Repeat 25 times*
45C for 10min
80C for 10min
10C hold

Our default is 25 cycles. You can reduce the number of cycles (we often do 15) to shorten the reaction time, but efficiency goes down. We’ve never tried doing more than 25 cycles

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