Sample Preparation Guide

 

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Sample Preparation Guide
 
1) Template preparation

The success of automated sequencing critically depends on having high purity template in the correct concentration. In case of samples in plates, constant concentration among wells is also important for the success of sequencings.

   i) Plasmid DNA

Preparation

There are many commercial kits available. Please submit DNA in deionized water. Do not use TE to dilute or resuspend the DNA because EDTA inhibits the cycle of sequencing reaction. We recommend using Qiagen miniprep/midiprep since both methods yield consistent purity of plasmid DNA for sequencing.

Please provide DNA in the concentration range of 100ng/µl and in the amount of at least 2 µg. Extra amount of DNA ensures that we have enough sample for a re-sequencing in case the first reaction fails. If samples' concentrations do not fall within this range or if you fail to provide us enough template to do the reaction, the experiment might be delayed.


   ii) PCR fragments

Preparation

The DNA is free of contaminants, unused primers or dNTPs. PCR templates that do not undergo any kind of post PCR clean up are not suitable for sequencing and will yield unusable sequence data. It is highly recommended that your PCR template is first observed on a gel to confirm that there is a specific product with the correct size. The Qiagen Gel extraction kit or PCR cleanup kit can be used to remove all of the unwanted elements from your template.


2) Host strains

The host strain can have an impact on the quality of the template DNA prepared even using the best methods.
DH5-α strains consistently produce good results. HB101, XL-1 Blue, JM109 and MV1190 are usually fine but JM101 is often poor.
The growth media you use can also affect the outcome yields, while LB is usually fine.


3) Quantitation

Sequencers are able to handle a wide range of DNA concentrations however with very low amounts of DNA the data quality will be significantly affected.
Using UV absorbance to quantitate dilute DNA solutions tends to give widely inaccurate results.
A good way to quantitate DNA is to run an aliquot on a minigel and compare the intensity to the a control of a known concentration. There are also concentration ladders that are commercially available. For each reaction, please provide 10 ng/100 bases, and at least 20 ng/µl solution in deionized water. Please provide at least 10 µl for any possible re-sequencing.

Measurement with electrophoresis is recommended rather than spectrophotometer-based measurements.


4) Primers preparation

Primer Considerations

Primers should be provided in DI water at the required concentration (see table above).

- High Purity
- Appropriate concentration
- No secondary priming sites
- No mismatches
- A length of 18-25 bases.
- GC% content between 40% and 60%.
- A Tm (melting temperature) between 55°C and 60°C
- No significant hairpins (>3bp)
- Free of salts, EDTA, or other contaminants

Please supply primers at concentration of (10 pmole/µl =60 ng/µl) in deionized water at volume of greater than 20 µl.

Primers supplied by customers should be desalted or purified. Crude primers generally do not work well for sequencing. We have the following primers available at no extra charge.

Go to Universal Primer List