Assembly Ladders
The Registry created a unique ladder to use with the various gels we run for assemblies. The ladder contains bands at the 4 most useful sizes, which are made by PCR. The detailed protocol for ladder preparation is available here.
Ladder Components
The ladder contains the following parts:
| Part | Plasmid | Primers Used | Size after PCR |
|---|---|---|---|
| pSB1AK3 | - | Prefix-R and Suffix-F | 3189 bp |
| I5010 | pSB3K3 | Prefix-F and Suffix-R | 998 bp |
| I13027 | pSB1A2 | Prefix-F and Suffix-R | 506 bp |
| J32015 | pSB1AK3 | Prefix-F and Suffix-R | 106 bp |
Versions
The ladder can be made in two different ways - so that the 4 bands are all the same molarity or all the same brightness. For assemblies, the Registry uses the equality molarity ladder, which each band at .9 GP/μL. By loading 20μL, the amount of DNA in each band is equal to the theoretical yield loaded from each assembly. This allows us to quickly judge the efficiency of the assembly.