endonuclease contamination in mini-preps - (Feb/12/2009 )

I'm working with the ligation of an 18kb vector fragment generated from a 20kb plasmid and a 1.8kb insert fragment generated from a 6kb and it's apparent that there's probably some non-specific endonuclease contamination and feel it may be negatively affecting my ligation attempts. I've done the procedure all in the same day, both are double digests, ran on gel & gel purified -> ligated & transformed into NovaBlue using a wide range of vector to insert ratios. As far as handling the endonuclease contamination are there any additional step(s) to a mini-prep or alternative DNA extraction method I can use?

Only because I'm doing some minipreps today on low copy- plasmids... (plus I like to be helpful ;-) )

The qiagen miniprep kit says you should do an extra wash with their PB buffer (no detail from them what the buffer actually contains, tho') to remove endonucleases.

You could also do another EtOH precipitation afterwards, or put your purified DNA back onto a miniprep column and re-do the protocol.

Hello

I doubt if the reason is endoculease contamination, the ligation that u r trying is tricky as the vector size is huge. I would suggest you to CIP the digests, precipitate and directly use for ligation, without running on the gel or column purifying. You will get a lot of religations, but screen a lot of colonies and u would surely get a positive clone. Thats what I do for ligations which involve large vectors.

Hope it helps.

TC

No mysteries. PB buffer is

* 5 M Gu-HCl
* 30% ethanol

See this page for the Qiagen buffer preps and a link to the Qiagen patent describing these in detail:
http://openwetware.org/wiki/Miniprep/Qiagen_kit

And make sure that you are not trashing your DNA with short wave UV exposure during the gel purification. Long fragments are more sensitive.

Thanks for the input guys,


Swanny - I do include the PB wash for nucleases, would you recommend doing it twice?

TC - hardcore man. I don't think my PI would agree to that but it works it works right?

phage434 - so using a UV gel viewer to excise out the bands on the gel would do it?

Well

In my lab we work woth proteins with sizes ranging from 200-400 kDa. Cloning them is very tricky specially at the final stages. I found out that avoiding agarose and precipitating is the best way to clone them, atleast in my hands. You have to do it to believe it.

Just set up 2 parallel digestions, process one according to yr boss and precipitate the second one.
Best
TC

Hebron on Feb 21 2009, 01:28 AM said:

Hebron on Feb 12 2009, 07:53 PM said:

many kits have an extra step to wash out the endonuclease. That should work. Also try precipitating DNA after the kit.

Hebron on Feb 21 2009, 06:58 AM said:

No, once should do it. I'd agree with scolix's comments about an extra EtOH precipitation afterwards.

Having a second-thought moment:
What does the vector look like on a gel before you do the digests? If it's OK, then endonucleases aren't your problem...
How old is the vector DNA?

Are you sure both ends of the insert are happy? Try a quick and dirty ligation on the insert and see if you get a ladder, or at least a 3-mer sized band. If you only get a dimer, one of the enzymes is doing something screwy. Try fresh enzyme or enzyme from another company (I know, I know, there's no real reason for the same enzyme from two companies to have such different properties, but it has happened in our lab too often to ignore).
Ditto for the ligase. Treat some DNA ladder with ligase and make sure the ladder shifts up the gel.
What about the ligase buffer? I have taken to aliquotting it into 10ul in PCR tubes and only using the tube once or twice. I also make sure the ATP is resuspended well, which means it needs to be warm, not simply thawed out. Cloning success has increased enough to keep doing this.