Cloning Conundrum - (May/14/2009 )

Hey everybody,

I really need somebody's help on this issue

I have a 500 bp and 1000 bp promoter fragment that I want to clone into a PGL3-basic vector. The inserts have a Bgl II and SacI restriction sites on the ends and are PCR purified ( qiagen kit).
So I did a double digest of both the vector and the insert , ran it on a gel, purified the bands using montage gel extraction kit.

Did the ligation at a ratio of 3:1 and 6:1 with T4 DNA ligase O/N @ 4C. (negative control had double digested vector plus ligase but no insert)

Transformed them into competent DH5 alpha cells.

Plated on LB plates with 100ug/ml ampicillin.

Got nice juicy colonies on all my plates and no colonies on the negative control.

Picked 5 colonies from each plate and they all grew in LB media with 100ug/ml of amp.

Did alkaline lysis miniprep and the cut vector is present but there's no insert. ?????

( the enzyme digestions are working coz I tested them out individually with their optimized buffers and with the buffers i am using and ran it on a gel with the uncut vector)

I don't know how to explain this and I don't know wht to change to make it work

Did you add a few bases to either end of your fragment so that the RE's could bind?

Ihatecloning on May 14 2009, 09:30 AM said:

If u r getting religations of ur vector, it is likely that one of the enzymes that u hv used hasn't cut the vector properly. How long did u digest ur vector? Further, it wud be a nice idea if u cud use a vector backbone which already has some insert at the appropriate RE sites, so that u know that vector digestion is efficient.

I did add a few base pairs at the ends.

Thats the thing, I don't know if they are re-ligated vectors. I digest for an hour at 37C. I tested out the enzymes individually on the vector and ran it in a gel and all of them seem to be cutting in the buffer i used for the reaction.

what do you mean? The same vector with a different insert to use as a control ?

Ihatecloning on May 18 2009, 09:59 AM said:

yeah, if u have an insert at the required RE site. if u have one, u can take advantage of that. digestion for 1 hr. is for checking of ur clones or for preparing the cut vector? if it is latter, then I wud suggest that u increase the time to 4-5 hrs.

actually both the digestions are 1 hour. I could increase the time and see.

thats the problem i don't have a vector with an insert in those sites or I would have used that as a control

I did try phosphatase treatment just to make sure i wasn't getting any religations. And I didn't get any colonies on my plates except one. I did the mini prep on that colony and my DNA is degraded.. its a smear on the gel but i can see the cut vector... I am so lost.. what is going on. How does DNA get degraded by miniprep analysis ?!?!?!

Ihatecloning on May 20 2009, 11:06 AM said:

uh..oh....hey plz dont get disheartened! lets see.......was it a digested DNA that u loaded on the gel? in that case, did u by any chance use any unautoclaved stuff, MQ, tips or anything.......ur stock DNA may still b fine, its only the digest which might have gone bad! n if u have loaded undigested DNA, what do u mean by saying, u can see the cut vector??

hey so I figured out the contamination was from the RNase that we ordered. I stopped using that and I have an insert in that colony that I mentioned before.. Yay!!!!

now i am just doing a PEG prep to get more of the construct .. we'll see how that goes!!

thanks for the help