I am cloning a mouse gene under a tissue specific promoter. The vector has polyA tail. In this case is a full length cDNA of the gene necessary of just the CDS would suffice. I am working on PCR for full length cDNA for months now unsuccessfully.
Hi Kathan Munshi
if you want your vector to express the targeted gene you'll need the UTR to be present in your construction. In this condition, since CDS present only the coding sequences, you'll need to do it with the cDNA. what is the size of your target? higher than 10kb I understand your problem.
if you need help, you're welcome
all the best for this new year
fred
Hi Fred. Thanks for the answer. My cDNA is 2kb but the PCR is totally suboptimal and not giving desired result. Have tried changing primers polymerases DNA source. Nothing seems to work.
2kb, it's not very long...
ok, send me a mail ([email protected]) and if you want, give me some details, as the gene name, the species, your PCR protocol and I'll design for you primers to get a clean PCR (I'm used in primers designing, with Oligo7, whatever the species..).
fred
Another solution would be to just skip the PCR step and have your gene synthesized exactly as you would want it to be. It is much more cost effective than wasting weeks or months of time.
Michael J. Benedik Can you let me know some companies who would provide this service?
Just do a quick google search on gene synthesis. Many companies that do DNA synthesis for oligos also do whole gene synthesis.
Dear Kathan Munshi
for human or mouse gene you can:
1) Follow the suggestion of Michael J. Benedik which has the advantage that you can ask to generate also a codon optimized DNA fragment for expression in E.coli. In this way you will improve the possibility to obtain a good expression level.
Generally for this service i'm using Genscript wiich is quite fast and relativelly cheap and if you would like they can direclty subblone the GOI in an expression vector and send to you directly the expression clone.
We are doing it routinelly for the cloning of recombinants mabs /variable regions of heavy and light chain), However this could be relativelly expensive (500-1000 euro) i tihnk for a so long fragments (2kba?)
Otherwiese an alternative, probably cheaper appoach for mouse and human genes is direclty buy a Cdna (non optimized) clone to be used as template for your pcr
look to the following links:
https://www.origene.com/products/cdna-clones
https://www.genscript.com/orf-cdna-clones.html
in this case the price can change a lot from gene to gene.
good luck
Manuele
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