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

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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:
    1. Anchor (BstXI digested) to Annealed Top and Bottom Adapters with BstXI and SapI overhangs to SapI digested DNA (to be unzipped)
    2. 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):

Recognition Sequence:...CTCTTCNNNNN...
...GAGAAGNNNNN...

After cut:

...CTCTTCN NNNN...
...GAGAAGNNNN N...

After looking over the Sequence Map, the overhangs for EarI will be (for each cut):

  1. G, CGAT (top, bottom)
  2. C, GCGA
  3. C, GAAA

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.

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.

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