Hi all, These days, I tried to move an insert originally in the TA cloning vector (pGEM-T Easy, Promega) to my target plasmid vector. I used SacII and NotI to cut TA cloning vector, ran gel and gel purified the insert (about 2kb). Then I used Promega T4 Ligase to ligate this 2kb fragment to my target plasmid (about 12 kb) which was also cut by SacII and NotI and purified. After the ligation, I transformed by heat shock on One Shot TOP10 E coli bacteria and plated on LB ampicilin. The following day, I saw a decent number of white colonies showing up while negative control got no colony, suggesting my plates were effective. Then I picked up 20 white colonies for colony-PCR using my target plasmid specific primer and some gave the 2kb band, suggesting the presence of the insert. But the problem arose when I minipreped and digested the recombinant plasmid. I setup a digestion with SacII, which only cuts once in the vector so I should see a 14 kb band. However, what I saw was a 5 kb band, exactly the same size as the TA cloning vector with the insert. I send it for sanger sequencing and effectively I was still having the original TA cloning vector with the insert.
What is happening? When I gel purfied the 2kb insert, I was pretty sure that I did not carry over the 3kb 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? I even repeated the whole thing cutting the TA cloning vector so it destroys the replication origin and still got the same result.
Can anyone help me how to address this? I repeated it for 3 times and each time, I got the same results!--correct antibiotic resistance, correct colony-PCR but incorrect vector--so I am writing for help!!!! thanks a lot! Lara
What percentage of agarose are you using? You are probably getting some vector contamination while you are gel purifying. Depending on the abundances of these species (cut vector vs cut product vs supercoiled vector), I think you will get contamination, no matter how careful you are. Getting a clean cut trying at these sizes is quite difficult and need really good resolution on the gel. When you tried cutting the ori, did you then treat the cut DNA product with a phosphatase to prevent self ligation during your ligation step? You can try that as a first pass to see if this helps. But I think my solution below is a cleaner and easier way to clone your insert.
KS solution:
One easy solution is to pull out the insert using PCR instead of using restriction digestion. Once you have the PCR product, you can then dpnI treat the PCR product prior to purification, to remove the template DNA (your pGEM-T vector). Column or gel purify the pcr product, depending how clean it is. Then clone as you been doing before. Note, make sure to treat your vector with a phosphatase prior to cloning to prevent self ligation events. This should resolve the contamination issue you are seeing.
Hey Kyle, thanks for your answer. I was trying to avoid starting all over again using PCR instead of restriction enzymes because it is a time sensitive project but I guess you're right, it is the most doable solution. Thanks again!
I was thinking that I did the ligation controls and the only vector control had colonies but they were all pGEMT vector instead of my destination vector, which doesn't make any sense. Also since I was so paranoid did a only ligase control and had no colonies which confirms the reagents are not contaminated. Could it be that the restriction enzymes are contaminated with pGEMT? And since a 5 kb plasmid is easier to enter the bacteria by heat shock that a 14 kb plasmid, maybe que little contamination that is there is enough to win over during transformation. What do you think?
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