EST vs. FL cDNA? - (Nov/16/2007 )
I am trying todecide where to buy my cDNA clone from. I need a full length, I think. But I do not fully understand what the difference is between an EST and full-length cDNA. If I order an vector with an EST sequence, is that the whole sequence, just the CDS, or will I have to use a race-ready or elongase step to get what I need?
Thanks
Thanks
EST just means that the insert was just sequenced once from one side. The insert in the vector may be larger than the sequence available, but there's no way to find out whether it contains your full cDNA or not except by ordering the clone and sequencing it yourself. If you have the money, it's easier to buy a guaranteed full length clone, or alternatively you can look for a publication where it was used and ask it from the authors.
If you haven't already, you could isolate your sequence from uncloned cDNA rather than a clone. You can buy the cDNA or make it yourself from RNA, you just need to choose the tissue where your gene is most highly expressed and purchase that tissue.
you can sometimes obtain full length cDNA clones. A japanese group has done a full length cDNA cloning project. All clones are deposited into the IMAGE collection. Just recently I obtained a cDNA clone for my gene which we could not amplify from our cDNA. you can first search from IMAGE website (http://image.llnl.gov/image/IQ/bin/buildIQ?categoryChoice=clone~Clone) to see if there are any full length clones available for your gene, then order the clone from companies such as Open Biosystems.
Thanks for all of the suggestions. I am looking into isolating the mRNA from tissue. I have tried looking up other clones, but it seems that no matter where I go, even looking it up on IMAGE, I always run into a problem. Either I can only locate 1 of the 2 genes I need or there is a different name, gene id, accession, etc. that takes so much time to find and compare translations. I am still working on that too.
Does anyone have any suggestions about suppliers of either clones or tissues? This project has been delayed by the previous company who cannot seem to send me non-mutated or incompleete sequences, and my bosses would like to make a purchase with higher probability of success. Are there any companies I should look into because of excellent service and products or any I should stay away from for known problems and unhelpful tech. service?
Thanks again
Does anyone have any suggestions about suppliers of either clones or tissues? This project has been delayed by the previous company who cannot seem to send me non-mutated or incompleete sequences, and my bosses would like to make a purchase with higher probability of success. Are there any companies I should look into because of excellent service and products or any I should stay away from for known problems and unhelpful tech. service?
Thanks again
Clontech and Biochain will cover any tissue RNAs or cDNAs you may wish to purchase. Origene is quite reliable for the clones they sell but are often ridiculously expensive.
Does anyone have any suggestions about suppliers of either clones or tissues? This project has been delayed by the previous company who cannot seem to send me non-mutated or incompleete sequences, and my bosses would like to make a purchase with higher probability of success. Are there any companies I should look into because of excellent service and products or any I should stay away from for known problems and unhelpful tech. service?
Thanks again
Clontech and Biochain will cover any tissue RNAs or cDNAs you may wish to purchase. Origene is quite reliable for the clones they sell but are often ridiculously expensive.
Origene is the one I am having a little trouble with right now. I was actually doing some research yesterday on Clontech. I think I am leaning toward the Marathon cDNA because of what is supposed to be simpler procedure. It has the special Marathon vector that is supposed to help/compliment both 5' and 3' RACEs. Have you all tried this? I am sorry to ask so many questions, but I really do not have any one here that can help me in this area.
.
Thanks