I want to make cDNA of my gene with two restriction sites at its ends for cloning and over-expression studies.I found that most of cDNA , commercially available are GFP tagged. I am little bit worried to think that, it may be helpful for localization and other microscopic experiments but, it may not be good for protein-protein interaction studies as GFP may hinder for proper protein folding.Should I go for normal cDNA amplification and restriction site designing through PCR? Please give me suggestions
Hi Maynak,
I like to use the full-length cDNA and insert my restriction sites on both ends using PCR. This way, I can easily ligate the cDNA to my collection of expression vectors that have no tags, flag or ha or fluorescent markers. This way, from one PCR, you can generate all the clones you'll need at the same time by doing the ligation, transformation, selection, mini-prep, quality control, maxi prep in parallel.
A good source of cDNA is GE Dharmacon (Openbiosystem) that offer full-length MCG cDNA for a very low price (http://dharmacon.gelifesciences.com/mammalian-cdna/mgc-cdnas/).
Alternatively, you can always look on Addgene (https://www.addgene.org/) to see if there is a plasmid ready to go (glycerol stock are $65) and do your PCR on it if needed.
Best,
Nicolas
I agree with Nicolas,
Alternatively, if you can't find cDNA you can always amplify it from an RNA prep using RTPCR. There are dual kits out there that allow you to amplify cDNA of a target in one mix (RT + PCR). You just add in your mRNA targeting primers that are also flanked with your restriction enzyme sites.
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