Spec-ed/Loaded DNA inconsistency after digestion of mini-preps - (Mar/25/2007 )
Hello guys, it's my first post here. I was hoping you will be able to help with my cloning.
So I ligated some shRNA sequences into a lentiviral plasmid and cloned in E. coli. I mini-preped the DNA and got good quantities (around 500-1000 ng/ul as measured by spectrophotometry). In order to determine if I have the right size inserts, I did a double digestion (1ug of DNA) to cut the insert out and run a gel. This is where the problems begin:
1) When I digest my samples I see no DNA whatsoever on the gel - controls are working
2) If I run my mini-preps undigested, the DNA is there but the quantity is much less that 1ug (the intensity is more than 5 times less that the control). This doesnt make any sense, as the spec values tell me that there should be 1ug DNA there! (also the spec curve looked fine and the 260/280 was normal).
3) I tried digesting 10 times the amount of DNA (supposing that this would be about 1ug as I judged by the intensity of the bands) but when I run on the gel I saw no high bands, just a lot of smear further down.
So my question is basically...ummm...what on earth is happening??? 
I thought that maybe there is RNA contamination in the mini-preps (although there is RNase in the buffers u use), which would give a higher spec value when less DNA is actually there. Any other suggestions? I read on another thread here that phenol contamination can throw off the spec - and I believe the miniprep uses phenol (it's a modified Qiagen protocol, basically uses the same buffers but without collumns).
But then I wonder why this smaller amount of DNA dissappears after digestion. Is it just that the restriction enzymes are so much more than needed and completely chop off the DNA at very small pieces that I cant see?
(I cut with EcoRI and Acc65I for 3 hours at 37, which works for other people in the lab)
I am just wondering whether I should start over and from which stage. Do the mini-preps again? Clone again from the start? The thing is, when I run my minipreps undigested, the bands are all around the right size so I dont want to give up!
I think what im gonna do tomorrow is to chloroform extract some of my minipreps, wash with Ethanol, spec again and re-digest and run on gel.
Thanks a lot in advance!
I usually dont rely too much on spectrophotometer for DNA conc. as they are not always accurate.
I would suggest using more DNA for analytical digest of the lentivectors. Which cells are you using to grow the lenti vectors? We use stbl3 and they have exonuclease activity and my DNA is usually degraded if I do minipreps with columsn and even if i do a analytical digest, I get a smear and I struggle manytimes to find the digested product. One important thing, is the yield is never too high so i have to often use most of the miniprep DNA for the digest.
Maxi's are fine usually as the nuclease is removed in those columns.
make DNA from 5ml of miniprep culture and try to use as much as possible for analytical digest. Depnding on the cell type you are using to grow, think of using columns if necessary.
If the undigested DNA is is fine, try to run all undigested vectors along with a control one ( starting plasmid), if the insert is big enough, you might see a difference if you run the undigested vectors.
Good Luck !!!
I would suggest using more DNA for analytical digest of the lentivectors. Which cells are you using to grow the lenti vectors? We use stbl3 and they have exonuclease activity and my DNA is usually degraded if I do minipreps with columsn and even if i do a analytical digest, I get a smear and I struggle manytimes to find the digested product. One important thing, is the yield is never too high so i have to often use most of the miniprep DNA for the digest.
Maxi's are fine usually as the nuclease is removed in those columns.
make DNA from 5ml of miniprep culture and try to use as much as possible for analytical digest. Depnding on the cell type you are using to grow, think of using columns if necessary.
If the undigested DNA is is fine, try to run all undigested vectors along with a control one ( starting plasmid), if the insert is big enough, you might see a difference if you run the undigested vectors.
Good Luck !!!
Hello scolix, thanks for ur reply!
I am growing in Top10 cells, which people in my lab have been using to clone similar inserts in the same plasmid. The inserts are not of lentiviral nature themselves (no repeats etc), just shRNA sequences targetting genes of interest.
During the past days I tried out some things. I tried digesting 5 or 10 times the amount of DNA, using a variety of enzyme unit numbers as well. The result is the same. I can see no insert or plasmid, just smear down below. When I run the undigested samples though, I can see the plasmid! One thing to note is that even when I load 10x the DNA amount and run undigested, the band is still much weaker than the control band. So you are right to suggest using as much miniprep product as possible.
I also tried to do a chloroform extraction of the minipreps, this reduced the spec values but again when I digested I only saw smear on the gel.
I have now started minipreping and cloning again. Next time I will make a 5ml LB culture as u suggested (this time it was 3ml). And maybe tomorrow I'll give it a final go with digesting all the miniprep I have. What I still dont understand though is why digestion gives all this smear, and makes the linearized plasmid band dissappear - you cant have that much star activity can you?
I use a modified Qiagen miniprep protocol, without collumns. I am not sure how genomic DNA is supposed to be taken out with this protocol...My insert is about 60bp so yes maybe I can see a difference between undigested minipreps and controls. Ι will try that tomorrow using all available DNA, and then repeat with digestion. Thanks for your suggestions!