I performed two LR reactions to generate an expression vector to express in zebrafish (using the Tol2 system). I am very familiar with this since I generated a lot plasmids with this approach in the past. However, this time, both using the Tol2 pdestpA2 vector and the pdestTol2CG2 I got a lot of colonies: as a lot I mean that often they were fused each other. None of them, however, was correct: both using digestion and sequencing. From what I understood, the vector I purified from bacteria has the promoter (from the 5' entry), my gene of interest, but lack the polyA, they only have the first 50 base pairs of the polyA.
Can anyone help me on how to fix this issue? I would try to transform other E.coli strain, I used home made one (derived from commercially available TOP10 and made competent by us) so it could explain the high yield of transformation.
Any suggestion?
First, I would make sure my donor vector and destination vectors are what they are supposed to be by restriction analysis.
Second if your colonies are giving you different vectors from what you are supposed to be. Maybe some recombination is going on in your bacteria that is modifying your vector. In that case, I would use Stbl3 bacteria. Good luck.
I agree with the Daniel, I would move your recombination transformation into rec- bacteria. Generally I use Stbl3 or NEB Stable competent cells which work quite well. Also, it might help to grow them up at 30 degrees instead of 37 (both plate and broth), as this might decrease the amounts of recombination that is happening, especially if your destination vector is a viral backbone. If you grow it up at 30 degrees though, it might take a bit longer for the colonies to show on the plate, so allow up to 24 hours before discarding plates if they don't contain anything on them in the morning.
I'm not sure I 100% understand what you are isolating. Are you saying that you are achieving recombinant clones that have undergone LR recombination, but the plasmid is not intact, or are you still amplifying your original pENTR plasmid? If you are amplifying your original pENTR plasmid, then perhaps there are too many transformants on the plate. I would plate 20 ul on one plate, and 100 ul of the reaction on another plate. If there are too many transformants, it could be that they are degrading the antibiotics and causing other clones containing the original plasmid to still grow up and not be selected against, I hope that makes sense. Make sure that there are a few well spaced out colonies on the plate and grow those up at 30 degrees in broth as well.
Thank both of you for your answers. Concerning the entry e destination clones, I usually used them since few months ago, and they worked well. I may check digestion of course.
Answering to Juliette, I have a combination of the two scenarios you depicted: I had one colonies that in restriction looked like the middle entry vector with my gene, other strange colonies but I had also tre colonies that contained the promoter, my gene and only the first 30-50 base pairs of the poly-A, so it sounds like recombination occurred but something got wrong. I picked well isolated colonies but they were only a small number, the plate was covered by tons of colonies.
I will growth bacteria at 30 degree and let you know.
Thank you in advance.
Domenico
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