PCR using an enzyme mix - never done this before, how do I set it up? (Dec/11/2006 )
Hello,
I plan on PCRing a 7Kb gene. Since there is an Sph1 site right in the middle, I plan on running two seperate PCRs and linking the thing together later.
I would like to try this with a wild-type Taq as well as a PFu-like Taq named Phusion. Can I use an enzyme mix to ensure I don't get mutations? If so do I just make the PCR as I normally would, but just add this enzyme as well? Has anyone any experience in this?
"Pfu like taq name Phusion" What in the world does this even mean? I think the Phusion enzyme would be insulted at being called "like taq."
In any case, 7kb is easily amplifed with Phusion. I don't see the need to mix in "taq" or cutting the template in half. If you do cut the template in half, 3.5kb and 3.5kb... there is no need to use phusion. It would be like using a math PhD student to answer basic algebra. I would instead recommed KOD hifi from stratagene. Its a proof reading enzyme, has good yields and reasonably cheap.
As for the PCR condition, I think you need to elaborate on "What I would normally do." as standard operating procedure is actually quite different from person to person, lab to lab.
"Pfu like taq name Phusion" What in the world does this even mean? I think the Phusion enzyme would be insulted at being called "like taq."
This was funny. You're right.
Interestingly I started this before I read your reply.
I ran a gradient using phusion (100ul master mix divided into 10-10ul reactions) to find the right annealing temp for my primer pair.
Now that I found it I am re-PCRing using that temp and a 50ul reaction. Gel is running now.
I wish I knew you are familiar with phusion before I started. I tried to amplify all 7kb with phusion, but could not get it to work. I tried different annealing temps, extension times, DMSO, BSA, GC buffer, etc..
and could nto get it to go. How do you set up Phusion reactions with a long amplicon off of genomic DNA?
the template's quality is the most important thing.
The DNA template must be really good. the cleanest, and most intact that you can make it. I often test my template first by amplifying a locus within the genome with a set of primers that I know work.
The quantity of the product, determines if I use the genomic DNA as it is or clean it up some more.
If this option is unavailable to you, I'll make a set of primers to do a short PCR, around 1kb long using the genomic DNA.
The next important thing is the primer's design. You want an area with is just very sightly GC richer. Far away from nucleotide repeats, and sufficiently long/specific that the primer bind only once (ideally) in the genome.
Primers should not self anneal, form hair pins or form primer dimers with its partner.
All that done, load in the primers... find its melthing temperature. The annealing temp for phusion is tm+3 Celcius rather then the usual (tm - 4 Celsius). See if this reaction work... if not, drop the annealing temperature. I have droped as much as 6 Celsius (the tm was just too high). Set the extention time so it increases in a step wise fashion, getting longer every cycle.
If that doesn't work, you can add Mg ions and drop the extention temperature. See if that work. Could also add chemicals like KCl, glycerol and BSA.
If your sequence is GC rich use phusion's GC buffer. COuld add 5M Betain, DMSO
It is a bit of an art. But after awhile you sort of get the feel of what you need to do to get the reaction going.
Also note, as the PCR product is rather large, yields are low. So it is important that the primers are designed well to avoid severe misspriming problems... which will compete with your product for dNTP
But should problems arise with misspriming, you can gel purify your band, and use that as the template for a further round of PCR.
The worry for long range PCR is error in the sequence... though it seems (most of the time anyway) that it is a irrational fear. Very few errors do occur. And yes keep the number of cycles down, around 25. if yields are low, do more reaction. I have done 8 x 50ul reaction just to get enough PCR product to ligate.
thank you