DNA isolation:
- For plasmid DNA preparation, suitable quality for automated sequencing can usually be obtained if a commercially available kit is used properly. Some kits include Promega wizard PLUS SV Spin kits and QIAGEN products. Manual methods such as alkaline lysis “boil preps” usually do not yield sufficiently clean DNA for automated sequencing.
- For PCR products, the quality depends on the specificity of the PCR reaction. If the PCR product is very pure, one can use a PCR clean up kit to remove primers from the PCR product, or one can run PCR products on an agarose gel and purify the desired band. Qiagen and MinElute kits are recommended.
Measuring DNA concentration:
Accurate measurement of DNA concentration is critical for successful sequencing. A spectrophotometer generally is not sufficiently accurate.
The DNA resource center (L-432/POSC) has a Fluorometer for DNA quantification. It is available for all users at no charge.
Alternately, run the DNA sample on an agarose gel with a quantitative standard. Compare the intensities of the template band with the comparable bands in the standard lane and estimate the concentration of DNA. This method requires some practice!
After measuring the concentration, please use PCR quality sterile WATER to dilute the correct amount of the template following the Table below (see Prepare sample for DNA sequencing).
Please DO NOT use TE or other EDTA containing buffer and avoid adding any divalent cations (i.e. Ca, Mn, Mg).Prepare sample for DNA sequencing:
For DNA sequencing, place the following in a single 1.5 ml microfuge tube:
1. The correct amount of template DNA (use table).
2. The correct amount of One primer (use table).
3. Bring total volume to 13 microliters with sterile PCR quality water.
Suggested Amounts of template and primer:
Template |
Amount |
Primer[pmol] |
PCR Product |
|
|
100-200 bp |
1-3 ng |
3.4 pmol |
200-500 bp |
3-10 ng |
3.4 pmol |
500-1000 bp |
5-20 ng |
3.4 pmol |
1000-2000bp |
10-40 ng |
3.4 pmol |
>2000 bp |
40-100 ng |
3.4 pmol |
Single Stranded DNA M13 |
50-100 ng |
3.4 pmol |
Double Stranded DNA (plasmid) |
300-500 ng |
3.4 pmol |
cosmid, BAC |
0.5-1.0 microgram |
5-10 pmol |
Bacterial genomic DNA |
2-3 micrograms |
6-13 pmol |
Primer design: standard annealing temperature is 50 degrees Celsius. Primers with melting temperatures around 55 degrees Celsius generally produce good results.
Tube Labeling:
Label the SIDE of your 1.5 ml microfuge tubes with:
* Date
* PI Name
* Template Name (4 characters max.)
* Primer Name (4 characters max.)
Label the tube TOP
1, 2, 3, ...., N, in the order that the samples appear on the DNA sequencing order form.
Phone: (479) 575-4463
Fax: (479) 575-7139
E-mail: uadnalab@uark.edu
