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| PCR templates should be purified using a method that eliminates primer dimers, primers, nucleotides, and extraneous bands. Agarose gel purification, Montage® PCR Cleanup Kits, Centricon 100 columns, Wizard® PCR preps, and Qiagen PCR preps have all been used successfully. After purification, it is important that you verify the presence of only a single PCR product band on a gel. Accurate quantification of template concentration is especially important for sequencing PCR products. Absorbance on a spectrophotometer at 260nm may not give an accurate measurement of DNA concentration. We recommend running the sample alongside a mass ladder of known concentration on an agarose gel. We ask (but do not require) that you send a gel photograph of the purified sample, along with your samples so we can verify sample quality before performing the sequencing reactions. We can process samples submitted in all common microtube sizes, although we prefer that samples be submitted in 1.5 ml tubes or 96 well plates designed for PCR. Please don't send samples in regular microtiter plates (i.e., those designed for immunoassays). Those plates are brittle and sometimes crack during shipment, resulting in sample loss. Samples can be submitted in two ways: 1. Sample DNA and primer premixed in one tube total volume 10 ul. Primer final concentration should be 2.5 uM (2.5 picomoles/ul). 2. 10 ul of DNA sample in one tube and 10 ul of primer in a separate tube. The concentration of the primer sent separately should be 5 uM (5 picomoles/ul). Be sure to label each tube (preferably on both sides) with the sequence name listed on the submission sheet. Each tube with PCR products should contain 10 ul total volume, at a concentration of 4 ng per 100 bases of PCR product length. For example, to sequence a 1 kb PCR fragment, we would need at least 10 µl of the purified PCR product at 4 ng/µl (40 ng total). We recommend that PCR product concentrations be estimated by running a small known volume of the purified PCR product on an agarose gel adjacent to a DNA Mass Ladder of known concentration. Remember that for sequencing, only one primer (Forward or Reverse not both!) should be included with the sample or you will get a double sequence. The sample must be in water or Tris buffer (not TE)! |
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