Double-digesting my vector - should I see a 76bp band? (Oct/03/2005 )
I'm double-digesting a 2,790 bp vector with XbaI and XhoI (using the proper Promega buffer... double-digests using the same enzymes/buffer work with another vector). After digestion I should have two bands: the band I'm interested in should be 2,714bp and the MCS chunk that I don't need should be 76bp. My question is, should I be able to see the 76bp band on a 1.0% or 1.5% agarose gel? I loaded 10ug and I'm only seeing one intense band at around 2600-2700bp. I've repeated the digest twice already and keep seeing the same banding patterns... perhaps I should try sequential digestion. Since I'm not trying to isolate that band, I really don't care if I see it or not. I just want to make sure that I am gel-purifying a digested vector with the sticky ends that I am interested in.
I can't remember how many ug/uL are in my DNA ladder, but the 100bp band is clearly visible.
i wouldn't worry about the 76bp band not appearing if I were you; I think you have to load lots of DNA and you won't usually see it on 1% gel
maybe 1.5%; I typically use 1.5%-2% when trying to visualize something that tiny and even then it's usually really hard to see unless you load a ton
you could spend days and days trying to get a pic of your 76bp band, when it might be easier to just ligate it and do the transformation
how does your double digestion compare to a single-digestion control? If you run it on a high % gel and give it time for good separation you may see a slight difference
Hey, thanks for the quick reply!
I actually performed the ligations under the assumption that I wouldn't be able to differentiate the 76bp band or the difference between a single cut and double cut vector. As a result, I didn't do a single-cut control, but I may do that on a higher percentage gel if I continue having problems.
The host cells I'm using grow slow (roughly 2-3 days before you see colonies). It's been 2 days and I have zero colonies (I plated 10uL, 100uL, 250uL, and whatever remained from my transformations). I tried ligating two separate inserts into digested vector at room temp for 2 hours using NEB ligase and ligase buffer (contains ATP). The inserts and vector were digested with XbaI and XhoI. I tried 1:3 and 1:5 vector:insert ligations (with no insert controls). Those are relative ratios (volume:volume), because I was out of low mass standards to gel quantify and our damn spec was down at the time.
Anyways, this was my first attempt at ligations/transformations and I made the competent cells, did the ligations and transformations, and poured/streaked the plates all in one day (a Saturday no less). I'm sure I missed something tiny somewhere, but I meet with my advisor tomorrow to discuss things.
Thanks for the help!
what about your controls?
if you linearize your plasmid as a control for the gel, purify the band and ligate it back together...good control for the ligation reaction
another control, set up a transformation with just plain ol' vector; this will tell you if your comp cells are good
takes lots of tubes but saves lots of time in the long run
good luck, Hank
Good idea on those controls.
This is a key control, and -- if your experiment did fail -- where I'd look first. How did you make your cells competent?
Oops, forgot about this thread. Anyways, I used CaCl2 to make my cells competent. I transformed those cells with uncut plasmid and it worked beautifully.