I used the TOPO TA Cloning Kit for Sequencing to clone a desired gene into the pCR®4-TOPO® vector. I have been able to confirm the presence of my gene by colony PCR. I then extracted my plasmids using Qiagen Midi Prep Kit. I did quantify them so the extractions are pure. When I run them in an agarose gel (1%), you can see my isolated plasmid is super-coiled. I want it in linear form so I am just cutting at 1 site and I have tried using the following enzymes: NcoI, EagI, and EcoRI (none of which will cut my insert). All of these enzymes are failing to digest, whether I do it for 60 minutes, 90 minutes, or overnight. I should only have 1 band around 4.1kb but am getting these results instead. Can anyone help to solve the issue?
Addditional Information:
Digestion is done in thermocycler to ensure consistent temperature
Using 1uL NcoI and 1ug of DNA
I have tried several extraction kits: Qiagen Mini, Midi, Maxi and ZymoMini
Colony PCR are not always reliable. Did you check your isolated plasmids for positive insert?
But since the digestion sites are not working, it is better to repeat the cloning.
Hi Nicole Belanger,
As I can see in your photo, the plasmid is almost completely digested after 16h. The problem could be that you have isolated genomic DNA together with your plasmid. The contamination with genomic DNA can saturate the digestion.
If you don’t want to repeat the extraction, try to purify the plasmid from gel and repeat the digestion. Anyway, you should run the reaction in a lower % of agarose (around 0.6-0.7%), as you are expecting a 4.1 kb fragment and you don't have good resolution in this range with a 1% agarose gel.
Papri Basak I did run my isolated plasmids to see if they were positive for the insert. It came up negative but we thought this may be because the plasmid is super coiled that it was masking the amplification site.
First, you must confirm that your fragment has been inserted into the vector backbone. This problem is well solved, that is, the extracted positive plasmid and the empty vector are electrophoresed together, and the gel concentration is 0.5%. During this period, it should be observed that the empty vector migrates quickly. Or perform sanger sequencing. Secondly, there is only one band of the fully cut vector, and depending on your results, it does not rule out incomplete digestion. Third, the enzyme has a fixed-matched enzyme-cut buffer, I don't know if you have considered it.
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