http://www.meta-genome.net/methods/ 2010-09-08T15:11:11-04:00 http://www.meta-genome.net/methods/ http://www.meta-genome.net/methods/lib/images/favicon.ico text/html 2009-05-31T14:04:17-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=gene_expression:optimizing_primers_for_sybr_qpcr&rev=1243793057&do=diff SYBR green assays suffer from an important artifact. Fluorescent detection cannot distinguish between the formation of PCR product and unspecific DNA synthesis. The most commonly observed unspecific PCR artifact is ‘primer dimer’, which results from self annealing and priming of primers during the annealing step of PCR. The following figure illustrates and example of primer dimer often observed in PCR reaction. text/html 2009-05-31T13:53:09-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=start&rev=1243792389&do=diff This site contains some common methods/protocols in microbial ecology. The material originates from many different places and some of it may be taken directly from other sources. I try to give credit where I can, but don't always explicitly state my source. So please use caution. The information is not intended for distribution and was written as a document for internal use only. text/html 2009-03-17T15:32:56-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=gene_expression:rna_electrophoresis_formaldehyde_gel&rev=1237318376&do=diff NOTE: Formaldehyde is toxic and should be treated with care. At 0.1 ppm or higher concentrations in air, formaldehyde can irritate eyes and airways. Inhalation can cause headaches or a burning sensation to the throat, and can trigger asthma symptoms. The EPA classifies formaldehyde as a probably human carcinogen. Some countries ban the use of formaldehyde in certain consumer products, such as cosmetics. As a results, use dedicated formaldehyde glassware for all solutions containing formalde… text/html 2009-03-02T17:15:52-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=cloning:commonly_used_primers_and_probes&rev=1236032152&do=diff Small Subunit Ribosomal RNA genes (16S) As in: Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp. 115-175. 27F 5' AGA GTT TGA TCM TGG CTC AG 3' eubacteria 357F 5' CTC CTA CGG GAG GCA GCA G 3' eubacteria ANTIgamma 5' CTC CTA CGG GAG GCA GCA GT 3' eubacteria (very similar to 357F) 530F 5' GTG CCA GCM GCC GCG G 3' eubacteria / archaebacteria 926F 5' AAA CTY AAA KGA ATT GAC GG 3' … text/html 2009-02-09T11:18:09-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=community_analsyis:trflp&rev=1234196289&do=diff What you need before you start * one or more four-bp cutting restruction enzymes (e.g. Alu, MnlI, RsaI, Sau3A) * pellet pait * prepare 8 μl pellet paint as follows: 6 μl glycogen 2 μl AB Loading Dye (no salt) * 3 M NaAcetate pH 5.2 * dilute this 1:4 * 95% ethanol (use the molecular grade ethanol) * 70% ethanol text/html 2009-01-26T10:50:55-04:00 Boris Wawrik http://www.meta-genome.net/methods/doku.php?id=gene_expression:nuclease_qc_assay_for_taqman_qpcr_probes&rev=1232985055&do=diff (notes from IDT Research Dept. 1/26/07) After the probe has been ordered, an easy test that can be performed to ensure that the probe is quenching properly is to read the fluorescence from an aliquot of a probe, then perform a nuclease digestion of that aliquot and take a second fluorescence reading.