User:Anthony Salvagno/Notebook/Research/2009/09/10/Gel of Sap Capped and other things

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I am going to run a gel of the Ligation from last night, digested pBS with SapI, and SapCap against a 1kb ladder and a 100bp ladder. The layout is as follows:

Lane Number. In Lane

  1. 1kb ladder
  2. 100bp ladder
  3. SapCapped (my clever name for the SappBS + SapCap ligation product)
  4. SapCap
  5. SappBS (I'm trying to think of other names for this. I've got:SapBS, SpBS, SBS)

Image to come (really this time). I'm turning over a new leaf where I write everything out in my notebook and actually finish things that I say I will do later.

Anyways, the idea is that I will see if I have a Capped Sap. There was a problem when loading, I doubt there will be anything in Lane 5 because I could only get maybe .5ul. I think there weren't actually 30uL of elution yesterday and that using 25uL was all of it. Hopefully I will see something so that I can see an elevated band when comparing SapCapped with SBS. We'll see.

While the gel is running, I will Qiaquick cleanup the SapCapped just in case it did work.

Results


This is very bad news indeed. Lane 3 is SapCapped and as you can see I got a buttload of concatamers. How can I stop this? Lane 5 is the digested plasmid which is good news that it digested well, and that the amount I had in the tube was enough to show up on a gel. Hooray for less than 1ul! Anyways, concatamers bad.

Solutions?

Well from the looks of it, we did get instances of successful capping because you can see a band slightly above the 3kb line. However I don't believe for a second that that band is 2.9kb + 15bp. What to do:

  • Next Ligation add way more SapCap. Basically overload it so there is no chance for religation.

Outside of that I wouldn't know what to do. This is an interesting development indeed.

Steve Koch 22:10, 10 September 2009 (EDT): Bummer! Lane 3 is pretty confusing. The first concatamer should be just under 6 kB, but there is a band right around 4 kb. Is this just circularized plasmid (nicked version)? Or is it a supercoiled doulbly-circularized fragment? Trying to look at your previous gel (http://openwetware.org/wiki/User:Anthony_Salvagno/Notebook/Research/2009/05/05/Gels) and the incomplete SapBS digestion, but still confusing. Anyway, I agree that the cap didn't work. The only things I can think are:

  • Cap is not phosphorylated?
  • We screwed up the overhang?
  • Cap annealing did not work well
  • Cutting pBS with SapI for too long resulted in lots of star activity which created self-complementary overhangs

Steve Koch 22:10, 10 September 2009 (EDT): I am wondering if we should abandon pBS temporarily and go with EarI on pBR322?

Steve Koch 22:17, 10 September 2009 (EDT): I see from NEB that SapI is not recommended for longer than 1 hour digestion. So, you'd be better off using more enzyme and less time. However, I don't think that necessarily means that long reaction would have had extra star activity. Check out the FAQs on SapI, though: http://www.neb.com/nebecomm/products/faqproductR0569.asp#726 Maybe the mixing is the reason you've been having trouble getting complete digestion.

Bad News

I've been trying to get my car insurance taken care of for a couple of days now and I absolutely need to get it done today. So I have to leave early to access information on my laptop (which is home) that I didn't save, but did not close out either. Hopefully everything works out well. Also I will not be in tomorrow because I will be on a plane to Orlando.

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