On behalf of the People’s Front of the Laboratory (or is that the Laboratory People’s Front?), I reproduce here the lovely response I had recently to a PCR whinge of mine, as it is information of potential use to many a ‘white coalface worker’, and is more likely to come up on a search in an actual post than languishing in a comments thread.
If PCR problems are of no concern to you, then be aware that Heather is a damn fine read regardless.
No harm, and no loss. PCR fine-tuning is an art. I have gleaned a number of tips cut and pasted below if you’re patient – you can see if you’re interested in trying any of them. The ones that have worked for us at various times are in boldface. Not all sources are appropriately attributed; it’s lab cookbook fare and you can usually find bits cut and pasted directly from other sites with a simple Google search.
The fact that I’m feeling slightly inhibited about uploading this here means that this is truly a social networking site.
But here goes:
1. [Reportedly glycerol (5-10%)], formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). Glycerol may protect Taq against heat damage, although plenty is provided in the Taq you buy commercially, while formamide may “relax” your nucleic acids.
2. 0.5 to 2M betaine (stock solution, 5M) is also useful for PCR with high GC content and/or long stretches of DNA . Perform a titration to determine optimum concentration (1.3 M recommended). Reduce melting temperature (92-93 °C) and annealing temperature (1-2°C lower). It may be useful to use betaine in combination with other reagents like 5 DMSO. Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits. (eg. GC-rich kits!) (11.7% [wt/vol] reportedly helps with hemoglobin/lactoferrin contamination of blood extractions.)
3. Bovine serum albumin (up to 0.8 µg/µl) can also improve efficiency of a PCR reaction. (0.4 µg/µl reportedly helps with hemoglobin/lactoferrin contamination of blood extractions).
4. See also Tavi’s PCR guide.
5. Higher concentrations of MgCl2 can be used to improve efficiency.
6. This buffer (1x) can work better than the buffer supplied from commercial sources.
- 16.6 mM ammonium sulfate
- 67.7 mM Tris-HCl, pH 8.89
- 10 mM beta-mercaptoethanol
- 170 micrograms/ml BSA
- 1.5-3 mM MgCl2
We’ve successfully used a variant of the above:
- 16.6 mM ammonium sulfate
- 67.0 mM Tris-HCl, pH 8.8
- 10 mM β-mercaptoethanol
- 6.7 mM MgCl2
- This will need to be aliquoted and frozen, as the β-ME evaporates off.
awww, * blush *
Heather! In RT-PCR and at the RNA extraction step, I am trying to work out whether I can leave the ol’ mercaptoethanol behind, which is nasty stuff. That’s what my Qiagen kit says…I am giving it a shot.
Shall let you know what happens. Have you tried?
Be interested to hear – but I haven’t tried it, except that when it’s gone from the PCR buffer mentioned above, said buffer no longer works very well. I thought b-ME was useful because it inactivates RNases (as mentioned here) by cleaving disulfide bonds, yet it doesn’t seem to perturb DNA polymerases. I don’t know about reverse transcriptase.
It’s pretty stinky but I don’t know if in the quantities at which we detect it away from the fume hood, if it’s really so bad as that. The odor threshold is reportedly between 0.7 and 35 parts per billion 1. Wikipedia even points to a couple of old references supporting a beneficial effect on mouse longevity (presumably because of its antioxidant activity).
There are vastly more experienced molecular biologists out there. It’s just when you’ve had to do troubleshooting yourself, you build up a certain folk wisdom. I hope other wise old folks will chime in.
K. Verschueren, Handbook of Environmental Data on Organic Chemicals, Van Nostrand-Reindhold, New York, 1977.
A great example of scientific networking (as opposed to notworking) – see this post about it
I had one go already and I was not satisfied with the answer I got (not conclusive). I am just about to relocate my super-clean RT-PCR area. Will run it again soon, as soon as I unpack my super-dooper new thermocycler and centrifuge. Yay!
Meanwhile, thank you very much.
As I wrote re: Maxine’s comment, the examples abound at Protocol Online and the Science Advisory Board forums, just to name a couple of sites. In case anyone comes across this and would like to look at some other points of view. Just keep on the lookout for bad advice.
Could try DTT instead; it is supplied in the kit with the Reverse Transcriptase I use, presumably for similar reasons. I think it may also keep working conditions sweet for the RT itself (which is, after all, a DNA polymerase); I’ve never left DTT out to test this – works fine with. See here for more. And it doesn’t stink as bad!
This brings back memories. In grad school, I had to do tons and tons of PCR on HSV DNA, which is 75-80% GC (awfulness beyond belief). I did try betaine, but without much luck. DMSO was always quite helpful, though I understand it can lower the fidelity of some polymerases. The new wave of polymerases on the market are getting really good at amplifying really difficult stretches of DNA – Phusion comes to mind.
Thanks for reposting this, Lee. It was indeed an impressive comment by Heather.