CDS CLONING QUERY
Posted 27 July 2012 - 08:35 PM
One of my seniors asked a question to me , kindly consider telling me your openion. I have cloned CDS of my gene of interest into pCDNA3 vector. The thing is that the region i cloned included almost 98% of CDS region but it does not include ATG codon. I also checked through qrt-pcr, and the construct is working fine. The question is that, is it fine to clone the cds excluding the initiation codon?
Please let me know your openion.
Posted 28 July 2012 - 01:31 AM
There are alternate start condons as well. Eukaryotes use them rarely, prokaryotes more often (e.g. lacI). If you did not include an ATG maybe there is one further downstream ...then you would have a truncated version of your gene.
What we are talking about? ...eukaryotic or prokaryotic systems?
Posted 28 July 2012 - 04:25 AM
I am worried if i could proceed to publish it or not, because someone might claim that i have not included initial ATG site, and if someone uses the primers i have used to check the over-expression, than i am not sure, what will be the probability of protein-over-expression. Please advise me.
i seriously hope i could use my results and constructs because i have seen some papers in which the primers they have mentioned (for CDS cloning) did not cover the initial ATG site.
Looking forward for the reply.
Posted 28 July 2012 - 05:12 AM
Next time i would spend more time on getting to know your expression vector before you start cloning. It is essential to know the precise features that are down and upstream your MCS.
Posted 28 July 2012 - 05:19 AM