Template:SBB-Protocols GGAssembly: Difference between revisions
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JCAnderson (talk | contribs) (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...) |
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Reaction mix: | Reaction mix: | ||
<pre> | <pre> | ||
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 | ||
6uL water | |||
10uL total volume | 10uL total volume | ||
</pre> | </pre> | ||
Latest revision as of 13:47, 29 January 2014
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
