C. elegans genomic DNA I've been using 300 ng as a template. Using rat brain cDNA as a template I amplified off of only 2 ng. However, this was only because I didn't have very much cDNA. If possible, it would be better to use ~200 ng of cDNA as a template, as Linda Buck did to amplify the odorant receptors.
Choice of templateGenomic DNA has the advantage that all members of your gene family are present in equimolar amounts, and genomic DNA is probably readily available. The obvious disadvantage is that introns may disrupt the primer sites, or may cause the amplification product to be so long that it is not amplified efficiently.
cDNA templates, though harder to obtain, overcome this problem. A big advantage of cDNA is that the desired amplification products should be of a known size, and you can therefore easily pick them out from among spurious products of other sizes. Remember that the "correct" sized band amplified off a cDNA template may be a complex mixture of products from many gene family members, so you may have to analyze many clones generated from such a band to assess its complexity. Linda Buck used random primed cDNA for her template, presumably to avoid biasing the cDNA towards the 3' ends of transcripts. In my case, I knew that the region I was amplifying should be at the extreme 3' end of the coding sequence, so I used oligo-dT primed cDNA.
For the lazy and rich, Clontech sells oligo-dT primed cDNA prepared from various tissues of many species to use as templates for PCR.
Analysis of PCR productsAfter amplification run 20 µl of each reaction out on an agarose gel. I use 2% 3:1 Nusieve:SeaKem LE agarose (you can buy this premixed from FMC) in 1X TAE. This gel is not very low melting, and thus isn't very suitable for cloning directly from the gel, but it gives very nice resolution. I use the 123 bp ladder from Gibco as a size standard. Obviously you expect to get products off the positive control, and not to get them off the negative control. Using the complex template, you will probably get a smear at the lower annealing temperatures, which will resolve into a small number of bands as the annealing temperature rises. I pick an annealing temperature that gives a modest number of bands, and then clone all these bands and sequence them.
If no products are evident in the experimental samples, a good trick to try is to use 2 µl of the apparently failed reaction as a template, and reamplify under the same conditions. This often gives visible products.
If you want to clone products that are only barely visible, you can get more of them by just reamplifying the original reaction as described above. Another way to amplify individual products separately is to cut the bands out of the gel that was used to analyze the original reactions (actually I take a bore out of the gel with a Pasteur pipette), melt the DNA containing agarose, and use 2 µl of it as a template to reamplify under the same conditions.
The above described method for reamplifying specific bands can (and should) be used to test amplified products to see if they are single primer artefacts. Use 2 µl of an agarose gel bore to set up each of three PCR reactions, containing either individual primer or both together. Obviously, you're only interested in products that require both primers in order to be amplified.
I clone PCR products by running the PCR reaction out on a low melt agarose gel (2% Nusieve agarose in 1X TAE). I cut the desired band out, melt it at 70·, mix well by pipetting up/down, and use 5 µl of the melted agarose directly in a ligation reaction with a dT tailed vector.
This vector DNA is prepared as follows: cut 1 µg bluescript SK with EcoRV in a 20 µl reaction. Add 20 µl 1X PCR buffer, and 2 µl 2 mM dTTP. Add 0.5 µl "ampliTaq" polymerase (2.45 U), and incubate at ~72· for 20 min. Run the DNA out on a 0.8% Seaplaque agarose gel in 1X TAE, cut out the band, melt at 70·, mix well, and use 5 µl in a ligation reaction. It turns out that only about 50% of the colonies obtained after transformation of this type of reaction may have inserts; the rest are vector reclosures. However, if blue/white selection is used, virtually all the white colonies have inserts.
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