|
Plasmid DNA Preparation
In order to obtain good sequence data the quality of the template DNA is extremely important. The following methodologies are recommended for preparing plasmid DNA for automated sequencing: Cesium chloride (CsCl) banding, Retrogen plasmid kit, Qiagen plasmid kit, Wizard plus plasmid kit, and other commercial kits available. Magnesium ions are essential for DNA polymerase activity. Template DNA and primer should be re-suspended in water or a buffer containing no more than 0.1 mM EDTA. The introduction of large amounts of EDTA in template DNA or primer will result in weak signals or short reads. The optimal concentration of the template should be at 0.2 µg/µl. PCR
Product Preparation
The purity of the PCR product is very crucial to obtaining good sequence data. Any PCR primers and/or dNTPs remaining in the PCR product will adversely affect the quality of the sequence data. If the PCR product has a unique band, it can be purified by the size exclusion method, such as PCR purification kit from Retrogen or Qiagen and Pharmacia offers. If the PCR product has more than one band, the PCR product should be run on the agarose gel in order to isolate the desired band needed for sequencing. The band can be purified by the Gel Extraction kit from Retrogen or Qiagen. The purified PCR products can then be quantitated either on the gel or spectrophotometer.
Recommended
DNA Template and Primer Quantities for Each
Reaction
| Type
of Template |
Amount |
Concentration |
| |
|
|
| PCR
product: |
|
|
100-500
bp |
150
ng |
20
ng/ul |
500-1000
bp |
200
ng |
20
ng/ul |
1000-2000
bp |
300
ng |
20
ng/ul |
| |
|
|
| Single
Stranded |
600
ng |
100
ng/ul |
| Double
Stranded |
1.5
ug |
200
ng/ul |
| Cosmid,
BAC, P1 |
4
ug |
200
ng/ul |
| Custom
Primer |
100
picomoles |
10
picomoles/ul |
|
