PCR amplificaiton isnt working - (Nov/11/2007 )
Ive been trying to subclone a 3,888 bp gene. Im trying to amplify the gene with RE overhangs and the PCR isnt working. The forward primer is a bit long, 51 bp (6 As and 6 bp restriction site...so 39 bp bind to the template). The reverse primer is 36 bp (6 As and 6 bp restriction site). According to IDT the Tm of the part of the primers that bind to the template are 58.6 and 58.7. The primers are so long because of the AT content (the GC content of the primers is pretty low maybe 40 %). Ive tried the PCR at 56, 55, 52 annealing temp. Ive tried increasing the annealing time from 30 to 45 sec. Ive tried lowering and raising the amount of DNA. All I see on the gel are a ladder of bands that are pretty faint. Im not sure what to do? Should I use another method to calculate the the Tm? Should I redesign primers that are shorter? But if I do then ill have a very low Tm like around the 40s. Any suggestions would be great.
You don't say why the AT content of the primers is high, but if it is because the template DNA AT content is high you may need to lower the extension (not the annealing) temperature to 64 or 66. More likely the primers have problems such as dimerization or internal self-priming. I usually redesign the primers after a few tries.
If you are going to re-design your primers you could consider making some of the bases LNA (Locked Nucleic Acid) bases. These are analogues of nucleotides, which increases the strength of the bonds and therefore the melting temperature. I first heard about them at a qPCR users meeting which I think was run by Stratagene? My memory is not best and I could only find information about them on the Sigma website:
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
Awesome Jess, cheers for that.
As for your problem Random, you said that you were sub-cloning, which makes it sounds as though you have the insert in another vector. If you do have it in another vector, can it be transfered across directly by excising the insert with restriction enzymes? That's generally a lot easier than amplifying it. Look for the same restriction sites in each vector to use for direct transfer, but also look at restriction sites that create the same compatible ends because they can be used too. Also look at if you can excise part of the insert. The rest of the insert can then be amplified later and ligated into the vector and the first part has been ligated in. If you are isolating the gene from cDNA or genomic DNA use more template to increase the amount of target for the primers to bind to.
If your stuck with the PCR though, I would definitely amplify the PCR product in two halves and join them together afterwards using splice overlap PCR. Amplifying a large fragment like that can be really difficult, especially if it doesn't work well the first time. A neat trick I like to do in my splice overlap PCRs is to design the overlap on top a restriction site, so if the splice overlap PCR doesn't work well, you can try a 3-way ligation to get the gene cloned. Just make sure the restriction enzyme is unique to the insert so the whole insert isn't digested (it can be in the vector, that doesn't matter) and that the overhangs it creates are not compatible with the overhangs on the outside of your insert. If you're extra tricky, look for a restriction site that is found between the two sites in your vector that you are using to clone the insert and then you can clone the two halves sequentially.
As for the primer design, personally I'd increase the Tm of your primers to around 70C. Higher Tm = more bases = more annealing (in general). Another thing that may make a difference is the 3` end of your primers. AT-rich sequences tend to "breathe" at the 3` end of a primer, meaning that the 3` end is not annealed well to the template and therefore does not extend well. Designing a GC clamp at the 3` end (3 out of the final 5 bases are G or C) of your primers helps to increase 3` annealing strength.
Good luck,
Rob
This may be a stupid question but what will lowering the extension temperature do?
As for your problem Random, you said that you were sub-cloning, which makes it sounds as though you have the insert in another vector. If you do have it in another vector, can it be transfered across directly by excising the insert with restriction enzymes? That's generally a lot easier than amplifying it. Look for the same restriction sites in each vector to use for direct transfer, but also look at restriction sites that create the same compatible ends because they can be used too. Also look at if you can excise part of the insert. The rest of the insert can then be amplified later and ligated into the vector and the first part has been ligated in. If you are isolating the gene from cDNA or genomic DNA use more template to increase the amount of target for the primers to bind to.
If your stuck with the PCR though, I would definitely amplify the PCR product in two halves and join them together afterwards using splice overlap PCR. Amplifying a large fragment like that can be really difficult, especially if it doesn't work well the first time. A neat trick I like to do in my splice overlap PCRs is to design the overlap on top a restriction site, so if the splice overlap PCR doesn't work well, you can try a 3-way ligation to get the gene cloned. Just make sure the restriction enzyme is unique to the insert so the whole insert isn't digested (it can be in the vector, that doesn't matter) and that the overhangs it creates are not compatible with the overhangs on the outside of your insert. If you're extra tricky, look for a restriction site that is found between the two sites in your vector that you are using to clone the insert and then you can clone the two halves sequentially.
As for the primer design, personally I'd increase the Tm of your primers to around 70C. Higher Tm = more bases = more annealing (in general). Another thing that may make a difference is the 3` end of your primers. AT-rich sequences tend to "breathe" at the 3` end of a primer, meaning that the 3` end is not annealed well to the template and therefore does not extend well. Designing a GC clamp at the 3` end (3 out of the final 5 bases are G or C) of your primers helps to increase 3` annealing strength.
Good luck,
Rob
I cant subclone it directly from one vector to another, I cant find the correct restriction sites. Ive actually got the last 3 of 5 bases are G/C on both the primers, including the last base being a G/C. Im afraid if I try and increase the Tm then the primer will be too long (its already 51 nt), would longer be ok? Im defiantly going to look into subcloning it in two halves.
http://www.sigmaaldrich.com/Brands/Sigma_G...LNA_Oligos.html
This journal may provide you with more information:
http://nar.oxfordjournals.org/cgi/content/full/gkl756v1
Cheers, Jess
Ill look into this, thanks.
just a question, which DNA polymerase are you using?
Pfu
Pfu
Ah well, that's not going to help. Good question pernese. Use Phusion polymerase. It's a protein fusion of Taq and Pfu so it has the robustness of Taq and the proof-reading activity of Pfu. It's easily the best cloning enzyme around and it's not expensive, I wouldn't use anything else. The problem with Pfu is that it isn't very robust, i.e. it doesn't amplify targets well, not as well as Taq or Phusion any way.
Primers above 50 bp are fine. You might get unlucky and catch one with an error in it but there's always a chance that is going to happen any way.