RT-PCR and RE cut site-will this work? - (Apr/24/2005 )
I am wondering if this will work. I have the mRNA of a cell that I will RT into cDNA. Then I'm going to ligate it into a pET-32a expression vector. I'm going to do HindIII digestion (recognizes AAGCTT) to make 5' sticky ends and ligate the cDNA ends to the pET vector. I'm using HindIII because I only want 1 cut site and this does that, and the pET-32a vector has this RE in it.
This is where the HindIII will cut my mRNA seq (4614 bp):
3181 acagggccca gaagctttag aggtatgaggtatgagg
This is where the HindIII will cut the pET-32a vector (5900 bp):
121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt cgacggagct
I'm wondering if this will work? The cut sites are not in frame with each other (ie:not in the same ORF), so is it possible to still do a proper ligation?
Also, I have PCR primers for my mRNA, but they are not near the cut site at all (they're between the 1000-2000bp)..which is good in a way, but wondering if that will negatively affect my PCR product if the RE cut site is very far away from the PCR primers? I plan on using one step RT-PCR.
When you digest a typical cDNA sample (generated using random or oligo-dT primers) with HindIII , you will generate 1000's of DNA fragments. You will have no problem ligating these into your plasmid but identifying the actual plasmid with your gene of interest will be the challenge. (note: this is how libraries are generated and you will require colony screening to find the plasmid with your gene).
Having the ORF out of frame is not important UNLESS this vector will be used for expression.
Why are you not PCRing your gene of interest first and then cloning? May take less time and will be easier to screen for.
Yes, I'm going to use this vector to express my gene. So I guess I can't do the method I described. I have to make the gene and vector ligate into the same ORF...that seems challenging.
I thought of taking the mRNA and using RT-PCR on it, so that the mRNA will make the cDNA and amplify from there.
I think still you should go ahead with PCR method to clone the gene of your interest in PET32a. There you would have nothing to worry on if you got the clone in cDNA library. cDNA library is goo option when you have to clone randomly genes of pathway, but if you know the gene and it's nucleotide sequence, always safe and easy to play with PCR method. You will have chance to put HindIII site to get the clone in a way you dream of.
Aru
Yes, I'm going to use this vector to express my gene. So I guess I can't do the method I described. I have to make the gene and vector ligate into the same ORF...that seems challenging.
I thought of taking the mRNA and using RT-PCR on it, so that the mRNA will make the cDNA and amplify from there.