Assembly Ladders

Revision as of 02:50, 6 September 2007 by SBurke (Talk | contribs)

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

Two variations of the Assembly Ladder

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.