Hi all.
It may sound a bit silly but I was wondering whether a SV40 polyA signal could be inverted.
For reasons, I have to introduce one in a plasmid of mine but I have only one unique restriction site available, meaning that it could get integrated either way. Any idea whether it'd impact its efficiency and mRNA stability in zebrafish ?
I've found on the web some clues that it may not matter much but I'd like to make sure.
Cheers.
I would sequence the clones and take the one with right orientation. Statistically this should not be so many, I would be more concerned regarding self ligation of the backbone without insert
I agree with @Ferenc Marincs, it would depend on what genes/promoters that you want to study and the purpose of your experiments. It's probably a good idea that you look at gene expression profiles at different cell densities (OD readings) including stationary cells, and under different growth conditions. There are a large body of published data in the field, for example, see The ISME Journal (2015) 9, 1130–1140; Nucleic Acids Research, 1999, Vol. 27, No. 19 3821–3835; THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 32, Issue of August 8, pp. 29837–29855, 2003. There are others. So, you may want to check the published studies to see if people already published on the gene/promoter that you would like to test, which would save you time and efforts. I think your gene/promoter of interest should be in those whole genome data files.
Dear Benoit,
You can easily solve your problem by amplifying the sv40 polyA from your vector template with a pair of primers having overhangs matching the arms of your acceptor plasmid in the right orientation. Then you can use a quick Gibson assembly protocol to ligate the pcr product with high efficiency.
If my explanation isn't clear enough I can help you with the design of primers.
Cheers,
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