I'm creating overexpression, N- and C-terminal protein GFP fusions, PROM.GFP, and PROM.GUS lines for my Arabidopsis gene of interest. I've been using classic gateway cloning.
The question is: Can we use gDNA instead of cDNA for creating the constructs?
When using cDNA, we often see one or multiple mistakes in the sequence after HiFi PCR. If we could use gDNA, the process would be less prone to errors, and it would be less time consuming.
The only thing I'm worried about is if the introns will still be spliced out properly, although I don't see any reason why they wouldn't.
Has anyone used gDNA for making overexpression of reporter lines in plants? Or what is your opinion about doing this?
Dear Schoenaers,
There is no issue using gDNA for Gateway cloning for any gene. Even I have used gDNA for cloning of few genes of Arabidopsis. The intron will be spliced out during processing of mRNA after transcription. One disadvantage using gDNA is that the cloning fragement will be bigger to clone. and sequencing will be costly. We use gDNA for cloning in some cases like less transcript of gene to clone using cDNA.
Good luck.
Great, that's encouraging Manoj. So you have experienced no problems with visualising protein or promotor GFP fusions? Have you ever checked for free GFP with western blot?
I agree with Manoj's answer. I would like to suggest that it is merely dependent on your experimental purpose. If you want to express this in Prokaryotes, then it is going to create problem because transcript will not processed there. If your (gDNA) gene of interest (GOI) is efficiently getting cloned in gateway vector then you can use it in eukaryotic system (efficient mRNA processing) for example you can study localization/co-localization of your interested protein in plant cells by fusing your GOI with fluorescent proteins.
Good Luck
There are no limitations in the gateway cloning technology. But, you must take care about the cloning strategy to achieve your goals such as destination vector, what kind of promotors have your destination vectors, LR reaction or BP reaction, intermediary vectors if is the case.
Regards.
Dear Schoenaers,
I have not got to GFP fusion protein. But i hope this will also work similar to normal expression of transgene cloned using gDNA. Using Gateway cloning there is no issue, since gene cassette will be arranged properly in destination vector. upto best of my knowledge there will be no such issue and u will get GFP tagged protein well.
Great, thanks for the advice everyone.
Further comments are still very welcome!
Hi Th Zheng,
That's indeed something to consider. I Have now repeated the HiFi PCR with several gDNA and cDNA stocks and will send them for sequencing to make sure we have at least one without mistakes. Thanks for the advice.
By using cDNA, I got little PCR product, not intense band in gel that I got after PCR using gDNA. Do you have any idea how can I get more PCR product from cDNA during Gateway Cloning? Manoj Kumar
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