It piles up again: passaging cultures and workload is suddenly multiplied, particularly when the last remaining Lab Manager / Technician is about to go on holiday; and the PCRs are still not working, but I’m homing in on that one; and counting more fixed, stained cells; and piqued by how reps are always wanting to talk to you, with their hairstyles and their suits and their big smiles, but when you’re trying to get a quote for a large order of a product you actually need quick, you can’t get a response; and wondering how it’s so easy to select the one lovely colony from one well and the one grotty colony from another that collectively depict the result you want to show, which makes one wonder at all those papers that show single colonies and have us believe that they are ‘typical’; and how desperate must be those fraudsters who take the same colony picture and Photoshop-turn it 90 degrees or whatever for another paper’s figure, and figure no-one will ever notice; and wondering what I’m going to blog about this week, because some days I can’t find my muse, nothing sparks (those who were in Scott Keir’s Unconference Session last weekend may understand); I mean, what’s the point if it bores people?… but then I walk into the Tissue Culture lab, and the student’s have got to the radio first, subjecting all to such phlegm-gargling twittering prattery that I actually feel better: if such purveyors of inanity can land salaries far fatter than mine, then what’s the harm, where’s the loss, in me firing off for zilch? So, I won’t feel too guilty about updating you on the PCR ghost; particularly as I reckon some of you might empathise and may like to read someone else whinge on what a drag lab work can be, especially when it’s backdropped by professional sycophants. So thank you, Nature Network, for allowing me to do this – and thanks for last Saturday.
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Playlist:
- Extreme: Saudades de Rock
- Living Colour: Live from CBGB’s
- Essential Soundtracks – the new movie collection
Lee Turnpenny 
Extreme: Saudades de Rock
Radio 1? Radio 1?
Clearly, some reprogramming is needed. Possibly with the back of a shovel.
I rather like Living Color’s album Vivid.
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:
Adjuvants –
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”http://www-lecb.ncifcrf.gov/%7Epnh/papers/TIBS/aug94.html . 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.
phlegm gargling
That will give me nightmares.
Nice to meet you at sciblog Lee.
Graham: Ditto (twice).
Heather: Cracked it! Not a case of tuning; more a complete halt of a previously working assay. After ruling out myself, it comes down to a reaction component; quickest to ditch the lot and start with fresh, which did the job.
But thanks for the tips – very kind response. I used to love playing around with these kind of variations when I was mad keen. I will keep by for when I hit problems with future assays. Cheers.
Good, Lee! After re-reading I thought so, but figured it may be useful for someone else. Then again, in the comments threads, not really (as they’re not searchable, grumble).
Then, damn it, Heather, lets make it so!