This is the gel pic that is confusing me and my boss - (Sep/05/2008 )
This is the pic, DNA ladder mix 5 µl, 2µ uncut vector, 2µl of single digest with Xho1 after two hours, 2µl single digest with Bgl2 after two hours, 2µl of old gel extracted double digest (the band I purified this from was thicker than this one!), 4x 10 µl of Xho1/Bgl2 double digest, 4x 10µl of Blg2/Xho1 double digest (5µg of vector, 20 units of enzyme, 100µl volume, in total 5 hours at 37 °C).
Stuffer sequence is supposed to be at around 1000, its supposed to be only one.
Would you say that the vector was double digested and use that band for a gel extraction? 
Vector is 7.3kb
By the way my boss solution was to perform another double digest (6 µg in 100µl) and then tell me to dilute that 1:10 and load a gel with that so the band wouldnt be so thick. She did this with a 2µl sample of the double digest and we saw a thin band at the right height.
Hi,
I'm not sure at all what you're talking about. What size fragments are you expecting from a single or double digest? Which bands are you interested in? What's the stuffer sequence you're talking about?
If you could give a bit more information, and tell us what it is that's confusing you, then we might be able to help you.
Ginger
Hehe youre right
Well the vector is a pSUPER.retro.puro with stuffer sequence. That means the vector is 7296bp long and when double-digest with Xho1/Bgl2 a approx 1000 bp stuffer sequence should come out.
Im interested in the vector sequence with Bgl2 and Xho1 overhangs. My problem is it looks as the the ratio between the vector that is supposedly double digest and the stuffer is gigantic, my boss says it looks like the vector isnt being completely digested and that the thick band that I get after the double digest is probably a mix of single and double digest vector.
Aparently separating the single digest (7.3 kb) and the double digest (6.3 kb) is tough. So as a control I ran uncut vector, single digests and my double digest to be able to see what the deal with the thick band is. Does it look double digested or like the digestion wasnt very efficient? And why would I get two stuffer bands? (1kb and 900bp). I digested for ages this time and it looked pretty much the same as when I double digested for 1hour . Except then I only got one stuffer band and the thick vector band at around 6-7 kb hihi
Ive ordered Xho1 from fermentas and I have Bgl2 from ferments, if it looks as though my digestion isnt working maybe I should try new enzymes, less material and longer digestion?
Or do you think that if I dilute those double digests that I have on the gel and let them run for ages on a 0.8% gel Ill succesfully get rid of what looks to be single digested vector?
Well the vector is a pSUPER.retro.puro with stuffer sequence. That means the vector is 7296bp long and when double-digest with Xho1/Bgl2 a approx 1000 bp stuffer sequence should come out.
Im interested in the vector sequence with Bgl2 and Xho1 overhangs. My problem is it looks as the the ratio between the vector that is supposedly double digest and the stuffer is gigantic, my boss says it looks like the vector isnt being completely digested and that the thick band that I get after the double digest is probably a mix of single and double digest vector.
This is probably true. First of all, it looks like your single digest with XhoI isn't working very well (you still have about 50% of your DNA in uncut, nicked form after 2 hours digestion.) I recommend you double-check your reaction conditions, and if everything's fine, order new XhoI.
Also given that your double-digest products are 1 kb and 6.3 kb, in a complete double digest you'd expect the intensity of the 6.3 kb band to be 6.3 times brighter than the 1 kb band because they'd be present in equivalent amounts. This doesn't seem to be the case. The big band is wayyyyyyyyyyy brighter than your small one.
I suspect problem is probably your XhoI.
No it's not. Make a 0.7% gel and run it out for a long time with single cut vector as a size reference to help you identify the double cut band. I do this all the time, and it's no more work than a regular gel purification.
Beats me. Are you sure your initial plasmid prep is pure?
Hope this helps,
Ginger
Thanx!
Yeah I think Xho1 isnt working, ill start of with less DNA 2µg or so and maybe use 3µl of Xho1 (10 units/µl) and 2 of Bgl2 in a 50 µl volume and cut for two hours.
Then ill run a 0.7% gel and see what happens.
Yeah I think Xho1 isnt working, ill start of with less DNA 2µg or so and maybe use 3µl of Xho1 (10 units/µl) and 2 of Bgl2 in a 50 µl volume and cut for two hours.
Then ill run a 0.7% gel and see what happens.
That's a lot of enzyme! Considering 1 u should cut 1 ug in 1 hour, that's 15 times as much XhoI as you should need! I normally cut 10µg DNA during that time in the same volume with much less enzyme (and remember, enzymes aren't cheap). If you do need that much XhoI you definitely have problems.
I know its a lot of enzyme, its really strange. I thought it was because I used Promega enzymes but then I had a similar problem with the Roche and Fermentas combo.
Maybe the maxi that I received isnt pure. I shyly asked when i received it if it had been sequenced but the PhD student said that it should be fine not to worry.
Im changing enzymes one more time, ill use Xho1 and Bgl2 from Fermentas and im going to ask if I can sequence the thing, it would make me feel better, I was once told that if I ever received a plasmid from anyone even if it was god I should have it sequenced.
Ive spent three weeks on this already and when I first started I didnt know whether I had the vector with the stuffer sequence or not which made it hard to know if the digest had worked.
Thank you for your help, Im going to take all you said into consideration and work on it!