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An adequate amount of the specific PCR fragment is needed in order to obtain satisfactory sequencing results. Judgments on the specificity and yield of the product can be obtained from the bands in the agarose gel after electrophoresis. There should be a single band in the sample lane. Extraneous bands will cause interfering background noise in the chromatogram. Occasionally primer dimer will be produced, presenting a band at approximately 50 bp. This should not present a problem if the band is weak. The volume recommendations listed in Table 5 of the product profile should be considered rough estimates. Each band in BioMarker® EXT DNA marker on the gel is at 50 ng, therefore if the sample band is equal in intensity to the marker, use 2 µl. If the band is weaker, use more. If the band is stronger, use less.
Glycerol is added to the template before drying, so as not to damage the DNA in the process. The samples can be dried at a temperature of up to 70°C. Due to the presence of glycerol, the samples will not dry completely. There will be approximately 1 µl remaining, perhaps a little less. This 10% extra volume will not interfere with the sequence reactions.
Scaling of the reaction mix saves time, and pipetting larger volumes increases accuracy. Distributing a master mix will result in a loss of volume, so that additional master mix is needed for a fixed number of reactions. The following is an example of scaling the mix for 48 samples. For this example, allow for 52 samples which is a 7.7% overage. The total volume of master mix will be 520 µl (10 µl/rxn x 52).
- Thaw reagents and keep on ice.
- Add the appropriate volume of PCR product into each well.
- Add 2 µl of 25% glycerol into each well with sample, then centrifuge briefly.
- Dry to approximately 1 µl.
- Combine reagents in order as described in Table 1.
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