Contamination with 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

Hi,
Sorry, don't really grasp completely what you're trying to describe.
Anyway...

A caveat, I think you should know too, is that TA cloning your insert will yield an insert DNA oriented in two probable directions.

E.g. -------------SacI===============>NotI--------------

or
------------SacI<================NotI-------------

ASSUMING that your ApaI is on the insert DNA (WILD GUESS ), and if it's near NotI, which is near SpeI too, you'll get the 5.3 kb fragment if you digested with ApaI and SpeI.

E.g. -------------SacI==============ApaI=>NotI--SpeI------------

or
------------SacI<=ApaI===============NotI--SpeI-----------


If it's being oriented in the opposite direction, it'll be away from Not I and when you're digesting with SpeI and ApaI, you'll get 1.5 kb and vector size.

Hope it helps.

Bests