Dear colleagues,
I try to insert 2 different PCR products (relatively the same size, apprx 3000 bp) to pCR 2.1.TOPO vector. For this purpose I add poly A to each PCR product. Consequently the PCR-poly a product was extracted from a gel and inserted into TOPO vector ( following the protocol, provided by the company)! For both inserts I got beautiful colonies. Minipreap was performed in parallel (see the attached file). After restriction analysis I found out that the PCR-2-TOPO construct is totally fine, but I do have problem with PCR-1-TOPO constrict. I always get very less plasmid with PCR-1 insert, although after restriction analysis I got bands with the correct size. I try to amplify the plasmid with MIDI preap- without success... I repeated the procedure 2 times- it is always the same!
My question is - what can be wrong with this construct?Why the TOPO Ecoli are not able to amplify it? At the same time, the other construct is totally fine.... the difference between both is only the insert, all the rest is the same....
I would appreciate any suggestions and recommendations!
Best wishes
Mariya
PCR-1 product is likely toxic, or at least mildly toxic such that you are able to get the correct colonies but the mild toxicity is preventing the full copy number of the plasmid in the host.
When you are attempting your midi-prep, are you inoculating your new culture with some of your overnight liquid culture? At the very least, you should dilute one of your correct minipreps to 100 pg or 10 pg per µl in Tris buffer, retransform, and inoculate the liquid culture from one of the colonies. If you see big fat colonies and small ones--and they're all reasonably separated--then the big fat colonies have likely kicked out or rearranged the insert and I wouldn't inoculate with one of those. I have come across this many times with a wide range of insert toxicities. Make sure you don't re-transform with too much plasmid, and if you want to transform with different masses of the miniprep DNA, then pick the transformation that used the lowest mass of DNA that you got colonies from.
Beyond that, other tips from the website:
https://www.thermofisher.com/order/catalog/product/K450002
Frequently asked questions (FAQs)
I’m able to see colonies on a plate, but when I pick them for liquid culture, no growth is observed. Why?
One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.
Suggestions:
1. Use TOP10F’ or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
Good luck.
Dear Christopher, thank you very much for the detailed answer! Actually, I never faced this problem before and I was also wondering whether is not a question of toxicity! I am going to follow your recommendations and I hope- I overcome this problem!
nice evening!
Mariya
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