Foreign band after double-digestion - (Oct/30/2005 )

Hi,

I've double-digested my 2796bp vector w/ XhoI and XbaI using a buffer that is appropriate for both enzymes (Promega Buffer D). Basically, I'm cutting out a 76bp band from this vector. I've done this before and have happily seen one band of the correct size. Lately, however, I've been getting a mysterious band at around 1500bp in addition to the band at ~2700bp. I've performed this on two separate preps of my plasmid, so I'm thinking that the issue is w/ my restriction enzymes. Anybody have any ideas before I try to get ahold of new enzymes? Because I get a band at ~2700bp and another at ~1500bp, and 2700bp +1500bp > 2796bp, should I just gel purify the band that looks the right size?

This is all a bit unsettling since I used these same enzymes to prepare my inserts and a single-cut vector for ligation control. The single-cut vector showed one band at ~2800, so that looked alright. My inserts looked fine as well. Maybe I should just try the ligations and see what grows..

By the way, I digested 20 ug of vector in a 60uL reaction for 2 hours w/ 2.5U of each enzyme at 37 C.

Thanks,
Hank

That's way too much DNA in your restriction digest. Normally, you should aim at 1 ug in a 50 ul reaction. You have about 20x that much. There isn't even, in principle, enough enzyme to digest that much DNA -- 1 unit digests 1 ug in 1 hour, under *optimal* conditions. Usually we use 2x to 10x this number of units. You have 2.5 units for 2 hours, so you could cut, optimally, 10 ug. Realistically, you can probably cut 3-5 ug.

You should have no reason to cut that much DNA for a ligation. Redo the reactions with 1 ug in 50 ul and run them on a gel.

Hi,

Sorry, that was a typo. I should have typed the last line as:
"By the way, I digested 20 ug of vector in a 60uL reaction for 2 hours w/ 2.5uL of each enzyme at 37 C."
2.5uL = 25 U of enzyme because it comes as 10 U/uL.

In any regard I was digesting that much because the Qiagen gel extraction kit that I've been using has been giving HORRIBLE yields (checked the pH of the extraction buffer and all that good stuff. I think the columns are bad). I just found a new kit though (Promega SV Wizard) so I'll try using that to purify serveral ug's of digested plasmid. That foreign band is still interesting nonetheless.

And when you say that you usually use 2x to 10x the number of units, are you talking about over-digestion? I noticed that in the NEB catalog is says that, when digesting w/ XhoI, you should do a 10-fold overdigestion. So does that mean I should use 10 times the amount of enzyme? Maybe it's just me, but if this were the case it seems like you'd have to massively scale-up your reaction volume in order to compensate for the amount of glycerol.

Thanks,
Hank

And when you say that you usually use 2x to 10x the number of units, are you talking about over-digestion? I noticed that in the NEB catalog is says that, when digesting w/ XhoI, you should do a 10-fold overdigestion. So does that mean I should use 10 times the amount of enzyme? Maybe it's just me, but if this were the case it seems like you'd have to massively scale-up your reaction volume in order to compensate for the amount of glycerol.

Yes, I'm talking about overdigestion, just as the NEB site suggests. Rather than scale up the volume, you should scale down the amount of DNA you are digesting. You should solve your gel extraction problem, rather than trying to compensate for it by overloading all of your reactions. I have consistently good luck with the Geneclean systems from QBio, but use whatever works.

Have you considered whether you really need to gel purify your digested DNA? Maybe you could just ligate and go. Gel purification has a whole set of problems, including exposure of the DNA to UV.

Hi,

That makes sense now, thanks.

I was going to gel purify my double-digested plasmid because I figured that the 76bp chunk I'm excising from the MCS will compete w/ my insert during the ligation. I basically wanted to limit the number of false positives.

I'm using guanosine to protect from UV damage.

After talking to a bunch of different people I feel as though I'm making this a bit too complicated. Does anyone actually still calculate the number of insert ends vs vector ends? Most people I've talked to just say "Yeah, I just use EtBr intensity to estimate the ratio of insert:vector". I went through w/ the calculations for the fun of it, but I just get the feeling that everyone does it a different way.

Thanks for the advice,
Hank