possible contamination of TA cloning vector? - (Sep/25/2006 )

Hi all,
These days, I tried to move an insert originally in the TA cloning vector (pCR2.1 Invitrogen) to my target plasmid vector. I used SacI and NotI to cut TA cloning vector, ran gel and gel purified the insert (about 1.5kb, which was well seperated from pCR2.1 vector itself after digestion). Then I used Promega Fast DNA ligation kit to ligate this 1.5kb fragment to my target plasmid which was also cut by SacI and NotI. After the ligation, I cleaned the DNA up and setup a kill digest using SacII (which has a cutting site in-between SacI and NotI sites on my target plasmid). I did transformation and screend for white colonies on LB+Cm25 plate with X-gal. The following day, I saw a decent number of white colonies showing up while negative control got no colony, suggesting my LB+Cm plates were effective. Then I picked up 20 white colonies for colony-PCR using my target plasmid specific primer (priming sites are close to SacI and NotI sites). All 20 colonies gave 1.5kb band, suggesting the presence of the insert.

But the problem arose when I minipreped and digested the recombinant plasmid. Since I need to move other fragment DNA into the recombinant plasmid via SpeI and ApaI sites, I setup a trial digestion with these two enzymes, whose sites are near to NotI site on the backbone. In theory, after digestion with SpeI and ApaI, the recombinant should give a about 5.3kb single band (because SpeI and ApaI sites are only 40bp away). But my digstion pattern showed that the 1.5kb insert was cut out!--exactly the same pattern as the insert was still in TA cloning vector!

I got puzzled-- when I gel purfied the 1.5kb insert, I was pretty sure that I did not carry over the 4.0kb TA clonig vector DNA (at least seen on the gel)--so how come it formed during the ligation? Is it due to the residule (tiny tiny amount) carry-over that was not visible in the gel? Since my target plasmid has different replication origion from pUC (the origion of TA cloning vector), I would bet the host e.coli may carry both plasmids. And TA cloning vector replicate in such a high copy number (around 300-500/chromosome) that it may mask the presece of my recombinat plasmid (copy number is around 20 per chromosome).--does this analysis make any sense? Or any other possible explainations?

How to address this? actually, I repeated it for 3 times and each time, I got the same results!--correct antibiotic resistance, correct colony-PCR but incorrect RE digestion patten--so I am writing for help!!!!

thanks a lot!

Paula

if I am understanding you correctly, what you are getting at the very end is your original TA+insert construct

you have tried to cut out this insert and shift it to another vector, but you keep getting the original construct

this may be a long shot, but the only thing I can think of is that you got partial digestion when you were cutting the original construct, with quite a bit of uncut supercoiled original construct masked by the 1.5kb insert band (there is variation, of course, but often supercoiled runs at roughly 1/2 the size of linear plasmid DNA on a gel)

so....there needs to be a control to ensure that the band you are getting is in fact the 1.5kb insert and not 1.5kb insert and supercoiled TA+insert construct. is there a digestion you can set up to completely rule this out? perhaps coupled with a higher-% agarose gel with a long,slow run time to make sure you're not picking up a tight doublet when you go to cut out your insert band? perhaps there's a third enzyme that cuts once somewhere in the TA backbone that you can use....do controls to make sure all the enzymes are cutting, and pick the 1.5kb from the other two bands? I see that you've done a 'kill cut' but perhaps it was not sufficient, or that any remaining uncut construct may still be there and will transform with excellent efficiency

are you with me? based on your gel pix, does this make sense? it is the only explanation I can think of, short of some bizarre recombination event that is highly favored. if you set up a ligation with a little vector, a little insert, and a little supercoiled uncut plasmid, it's a sure bet that your transformation will vastly favor the supercoiled uncut.

good luck! please let me know what happens

I am not completely sure I understand your problem, but I believe your problem is the following:

the TOPO 2.1 cloning vector has a SpeI site between SacI and NotI. So you subcloned the new SpeI site into your new vector together with your cDNA. The new SpeI site is close to the SacI site, so in the new vector you now have 2 SpeI sites flanking your insert and that is why you cut them out when you do the SpeI ApaI digest (but you were thinking that there was only the SpeI site close to the NotI site). Basically if I am correct, you will see that you will cut out your 1.5 kb insert with a SpeI digest alone, you don't need ApaI for that.

I hope this helps,

Vivian.

Hi, Vivian and Aimikins,
sorry for my long description of the problem which may confuse people--what Vivian suggested is exactly correct!!!! YOur eyes are sharp!

this morning, I setup a single digestion with another enzyme. ApaI. An around 5.0KB (my target plasmid is 3.8KB, the insert is 1.5kb) band is generated as seen by the gel, which suggested the recombinant plasmid is correct. I also found that the E.coli which carry this recombinant plasmid DOES NOT grow in LB+Kan plate, suggesting of absence of the TA vector in the cell. When I double checked the pCR2.1 map, I indeed found a SpeI site between the sites of SacI and NotI. So when my recombinant plasmid was cut with SpeI plus whatever enzyme, SpeI would cut twice to pop out the 1.5kb insert!

thanks guys! I am relieved now..... I will post another cloning design for the discussion in a moment!

cheers,

Paula

Cheers