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.