question about PCR clone a cDNA - (Sep/08/2005 )
Hi, I'm new in all of this of PCR, primer design, etc and I have a basic question.
I'm using NCBI and the access number gives me a mRNA sequence and the transcription gives me the protein sequence (245aminoacids) which is on the middle of mRNA sequence.
If I want to get the protein, then what I must do? This is what I would do:
- With the mRNA sequence design two primers one upstream (30-50 bp before) of the sequence of the protein and another donwstream.
- Then, perform RT-PCR to get the cDNA.
- Finally standard PCR with the designed primers and I will get the protein wanted in the gel which I must purify.
Am I right? What's your advice?
I hope I've been clear. Thanks
Always in training
sort of. You will get the coding sequence of the mRNA which is translated into the protein
In the mRNA file, where it says CDS start (under the mRNA start info) this will give you the info you are looking for. This is the start codon to stop codon, whereas the mRNA sequence contains 5' and 3' UTRs
- Finally standard PCR with the designed primers and I will get the protein wanted in the gel which I must purify.
I don't think you will get any protein by doing PCR, unless you invented a new type of polymerase
.To produce a protein, you will have to clone your cDNA in an expression vector and make protein in a mammalian cell or bacteria, depending on your vector.
Alternatively, you may clone your cDNA into a vector containaing e.g. a T3 or T7 promoter and perform in vitro transcription/translation
I don't think you will get any protein by doing PCR, unless you invented a new type of polymerase
.Sorry about the new protein polymerase (we are still working in this patent
) I made a mistake. What I meant is when I get the gel, then I will purify it to obtain the sequence searched, insert into a plasmid/vector and finally express this in the cell.
If anyone else have some extra advice, it will be welcome.
hi,
i want to quantify the mRNA with another technique than spectrometry (OD).
Are you aware of any alternative?
thanks. 
you can run a gel and do densitometry against a known standard
i want to quantify the mRNA with another technique than spectrometry (OD).
Are you aware of any alternative?
thanks.
You can use a flourometric assay with a dye that binds to mRNA, such as RiboGreen (molecular probes). Then you need to make a standard curve with a known mRNA concentration.
With a fluorometric assay you can detect even small amounts of mRNA, it's very sensitive.
k_josefin
thancks you! I go to look this...
Sacha.
I don't think you will get any protein by doing PCR, unless you invented a new type of polymerase
.Sorry about the new protein polymerase (we are still working in this patent
) I made a mistake. What I meant is when I get the gel, then I will purify it to obtain the sequence searched, insert into a plasmid/vector and finally express this in the cell.
If anyone else have some extra advice, it will be welcome.
HI To quantify mRNA...have u tried Ribogreen flourimetry? Ribogreen is a flourescent dye that binds only to RNA in your sample, and u can actually measure the intensity using a flourimeter, and amt of flourescence is directly proportional to the amount of RNA in your sample. Its very accurate andhelpful to know exact amounts for any downstream assays.