HPP Assembly Procedure

Edge Assembly

This assembly uses BsmBI to free up the Half Edge Word in the first and second Half Edges. Ligase drives the reaction toward completion. Equimolar amounts of first and second Half Edges are used. Reactions are scaled up 5 fold to provide enough product in 5 ul for use with 10 ng of destination vector.

To determine the 5X amount for use in this reaction, use this formula:

10 ng destination vector x (Half Edge plasmid size/2114 bp destination size) x (6)

9 ul dH2O
5 ul First Half Edge (5X Amount)
5 ul Second Half Edge (5X Amount)
2.5 ul 10X Promega Ligase Buffer
2.5 ul 500 mM NaCl
0.5 ul BsmBI
0.5 ul T4 DNA Ligase
Total 25 ul

50 cycles of 50C for 2 minutes/16C for 3 minutes
80C for 20 minutes
   

Use 5ul of this Edge Assembly for Graph Assembly