I am cloning a fragment into my expression vector using XbaI and HindIII digestion. I linearized my vector and did gel extraction to remove the excised fragment from the vector. (The vector was from another project, so had a 1kb fragment excised by the digest). My insert is a 2kb PCR product which I digested and then cleaned up on a pcr cleanup column (because the digest only removed 6-10 bases from the ends of the PCR product)
After ligation and transformation, I have many more colonies on my positive plate (vector + insert), but I still have quite a few on the negative control plate (vector only).
My question is, why are there ANY colonies on the negative control plate? My hypotheses are:
1. Some supercoiled, undigested vector was present along with the digested vector in the band I excised.
2. T4 ligase has some ability to ligate incompatible ends, thereby re-circularizing my vector.
3. I read in one posting that gel purification can actually blunt DNA containing sticky ends. The Internet does not seem to have other references to this phenomenon, though. But if that is true, the T4 ligase could achieve blunt end ligation of some blunted vector that was present.
4. Some linearized vector is getting transformed into the bacteria and somehow getting ligated, incorporated, or driving transcription of the resistance gene it carries before being destroyed by nucleases.
What is the correct answer?
Thank you!
-Eric
Hi Eric,
The only viable hypothesis is #1 which can contribute to seeing colonies on the negative control plate. You say the negative control plate is vector only. Is that vector with or without ligase? Vector without ligase controls for supercoiled DNA that is present. Vector with ligase controls for vector that is only digested by only one of the enzymes (XbaI or HindIII) which then can be ligated to itself. Without having both of these controls you will not be able to determine why you have so many colonies on your negative control plate. Theoretically you should not see any colonies on your negative control plates but in reality you will see a few/some colonies due to incomplete digestion of the vector.
Hi Eric,
The only viable hypothesis is #1 which can contribute to seeing colonies on the negative control plate. You say the negative control plate is vector only. Is that vector with or without ligase? Vector without ligase controls for supercoiled DNA that is present. Vector with ligase controls for vector that is only digested by only one of the enzymes (XbaI or HindIII) which then can be ligated to itself. Without having both of these controls you will not be able to determine why you have so many colonies on your negative control plate. Theoretically you should not see any colonies on your negative control plates but in reality you will see a few/some colonies due to incomplete digestion of the vector.
Hi Eric,
I also think that point 1 is the most likely one for colonies on the negative control plate, but there are some other possible reasons. If your vector is very large, you may removed the excised 1kb fragment not complete by gel extraction or even digestion was not complete. Another reason could be a contamination of any used components with a different plasmid. Any components used in ligation reaction, medium, agar plate or the bacteria itself could be a source of such a contamination. You need only a few vector copies to generate colonies on your plate. To be sure you have to analyse the vector sequence of the colonies of your negative plate. If you won’t find a vector in your negative colonies your antibiotic concentration is too low or you should use fresh antibiotic stocks.
Good points, Detlef. Next round I will also include a no vector control to control for potential contamination of reagents/equipment.
You can also phosphatase your vector, rendering it unable to relegate under any circumstances. This may help reduce the background. I agree with Tom on the importance of controls to understand what is going on.
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