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#1 Custom Gene Discovery |
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Submission Template Requirements
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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 purified plasmid DNA can then be quantitated either on the gel or by spectrophotometer. The optimal concentration of the purified plasmid 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 by spectrophotometer. The optimal concentration of the PCR template should be at 0.02 µg/µl.
QC Your Templates
It is very crucial that you need to measure the concentration of your template accurately, either on the agarose gel or by spectrophotometer. The #1 factor that causes poor sequencing results is due to inaccurate concentration. So, please verify the concentration of your template accurately.
Recommended DNA Template and Primer Quantities for Each Reaction
DNA and Primer Separated
DNA |
Concentration |
Amount |
2x Amount |
PCR product DNA |
5-20ng/µl |
50ng |
100ng |
Single stranded plasmid |
50-100ng/µl |
250ng |
500ng |
Double stranded plasmid |
50-200ng/µl |
500ng |
1000ng |
BAC |
100-200ng/µl |
2000ng |
4000ng |
Primer |
10pmol/µl |
5µl |
10µl |
We recommend 2x amount of DNA in case we need to repeat the reactions.
DNA and Primer Combined
DNA |
DNA |
Primer |
Total Vol. |
2X DNA |
2X Primer |
2X Total Vol. |
PCR product DNA |
50ng |
10pmoles |
10uL |
100ng |
20pmoles |
20uL |
Single stranded plasmid |
250ng |
10pmoles |
10uL |
500ng |
20pmoles |
20uL |
Double stranded plasmid |
500ng |
10pmoles |
10uL |
1000ng |
20pmoles |
20uL |
BAC |
2000ng |
10pmoles |
10uL |
4000ng |
20pmoles |
20uL |
We recommend 2x amount of DNA in case we need to repeat the reactions.
Example 1: If your PCR template and primer are at the concentration of 20ng/uL and 10pmol/uL, respectively, you need to add 2.5uL of PCR template and 1uL of primer to the 1.5mL epi tube, and add 6.5ul of H2O to bring the total volume of 10uL.
Example 2: If your Plasmid template and primer are at the concentration of 200ng/uL and 10pmol/uL, respectively, you need to add 2.5uL of PCR template and 1uL of primer to the 1.5mL epi tube, and add 6.5ul of H2O to bring the total volume of 10uL.
Single Tube Sequencing
Smaller orders should use 1.5mL epi tubes. Screw cap tubes are more time consuming so pop caps should be used. Each tube must be clearly labeled to match with the order forms. Tube strips while convenient are hard to label and keep track of so for large orders (24+ samples) use 96 well plates. We recommend using strip lids to cap plates as they prevent leakage and cross contamination – especially for plates shipped overnight.
96 Well Plate sequencing
Please submit your DNA and primers at the concentration according to the table above. The concentration of DNA and primers should be normalized and arrayed vertically from A1-H1, A2-H2…etc. on the 96 well plate. The plate should be sealed or capped using strip lids to prevent leakage and shipped overnight. |
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