HMK-Tagged PCR Cloning Problem... - (Jul/28/2012 )

I have a protein, which I need to place an HMK tag at the end of its N-terminus. The protein's sequence is already inside of an expression vector, pET-15b. I did PCR with primers containing the HMK sequence on this vector and isolated the product from a gel through phenol/phenol-chloroform-isoamyl extraction. I produced a rather good yeild as oppsoed to when I used the Biobasic Gel Extraction Kit which gave me no yield. I went ahead and did a double digest on my plasmid and this product with NdeI and BamHI. According to my gel, the vector cuts perfectly. If anything was a problem, I'd assume it would be the insert not cutting properly. I then, proceeded to do low salt ethanol precipitation and a ligation reaction.

The first time I did this, I got colonies on every plate. Even my negative controls. But for the sake of just seeing what I had, I did PCR on my different colonies. My PCR turned out fantastic! A bigger product exactly where I was expecting it to be using two sets of primers! I thought no way this is coincidence and I got lucky. I went ahead and then tried to do a restriction digest on a purified plasmid. However, it seemed to be very smeary and contaminated with genomic DNA. I said forget this and sent it for sequencing from a private company. They used the same primers I used, but said they could get no sequencing information, which I thought was weird.

I re-purified this plasmid and did PCR on it again. Again, it yielded the results I saw prior, but again, restriction digest looked horrible. Again with this smear. It can't be the purification, because I've done purifications plenty of times before and this didn't happen. So, I'm lost?

How did you purify the plasmid post picking the colonies? If you did a miniprep, you may have had some residual medium left behind, which means that you get some bacterial decay products (cell wall etc) left behind, that inhibit digests post purification.

It could be that the colonies have more than one form of the plasmid in there.

What controls did you have for the ligation?

bob1 on Sun Jul 29 00:08:16 2012 said:

Thank you so much for the response. 1) I purified the plasmid with Qiagen Maxi prep. 2) I think the digest was fine, because without the Restriction endonucleases you saw no digestion, but with the endonucleases you saw a smear suggesting this was probably as you said multiple plasmids in one bacteria or contamination with genomic DNA. For sure not RNA, because I did add RNase the second time around. 3) The ligation controls included: A) no ligase, plasmid, insert, insert, no plasmid, ligase, C) insert, no plasmid, no ligase, D) plasmid, no insert, ligase, E) plasmid, no insert, no ligase, F) no plasmid, no insert, ligase and G) Pet15B without digestion.

bob1 on Sun Jul 29 00:08:16 2012 said:

I should also add, I tried to do a transformation with the plasmid purified, that failed too!

Wow, you went to a maxi straight from colony picking? I would suggest that you do a miniprep in future, much much cheaper and a whole lot quicker.

Your ligation controls look fine, in fact you probably don't need the insert only ones. Unless the insert contains an origin of replication, it won't be amplified in the bacteria.

I think you will save time and money by repeating the process rather than trying to figure out the problem, it sounds like you have carry over of something from the prep, or some contaminating plasmid or insert (primer stocks or other component of PCR contaminated?).