User:Anthony Salvagno/Notebook/Research/2009/08/26/Tether Construct Ideas with Koch

Koch and I were spit-balling yesterday about how to make the construct more efficient. Here are some quick notes and I'll detail it more later.
 * The way it is now I ligate 2 ways:
 * Anchor (BstXI digested) to Annealed Top and Bottom Adapters with BstXI and SapI overhangs to SapI digested DNA (to be unzipped)
 * Anchor (BstXI digested) to Annealed HP adapter, A+A (as I call it) digested with NotI, A+A with NotI overhang ligated to NotI digested DNA
 * Neither option is uber-efficient, but I think the NotI technique is a little more fool proof than SapI


 * 1) Digest the SapI and NotI DNA with the other enzyme (digest with SapI on already digested NotI stuff) and ligate correct end to proper overhang
 * 2) Do everything with EarI

EarI Possibility
Circular Map and Sequence Map.

According to those links, EarI cuts 3 times. The recognition sequence for EarI is (Thanks thelabrat.com):

After looking over the Sequence Map, the overhangs for EarI will be (for each cut): So it looks like it would be ok to use EarI. Koch used pBR322 in past research and EarI cuts twice there (two of the overhangs from pBS are identical) and so I don't see any real difference between our two products.
 * G, CGAT (top, bottom)
 * C, GCGA
 * C, GAAA

Another circular map showing all digestions that we have done/could do in pBluescript. It looks like EarI flanks the MCS and is either identical to the SapI site or is very similar. Ok I just checked and SapI and EarI cut at the exact same location. I don't know if that is good or bad right now. I need to think.