- How to work out with a template containing prohibitive secondary structure?
Just notify us on DNA sequencing-work request form. We have proprietary protocols for those template with GC rich and secondary structure.
- What notes should I take of preparing PCR product for template?
- Check the specificity of product by gel electrophoresis.
- Remove dNTP and primers by purification before sequencing.
- Add only one strand of primer.
- Minimize the time of UV exposure when processing gel extraction.
- Decrease the volume of eluate when processing gel extraction.
- How do I determine the concentration of plasmid DNA?
- Check the DNA and RNA contamination by gel electrophoresis.
- Estimate the DNA concentration with a marker of known concentration.
- Determine the ratio of absorbance at 260nm and 280nm with a spectrophotometer
Unappropriated amount of DNA may cause sequencing to fail. Therefor, we have a guideline in page "Sample preparation".
- What notes should I take of primer design?
- Length of primer should be about 18-24 bases。
- Tm of primer should be above 45℃. However, the annealing temperature in our protocol is 50℃.
- Minimize the self-dimerization and prevent secondary structures in primer.
- Check the specificity of primer binding site.
- Why did I get a failed DNA sequencing?
Causes of failed DNA sequencing reactions:
- Poor quality DNA
- Degraded or failed synthesis primer
- Too much template DNA
- Wrong primer used
- Bad water
- Template DNA is contaminated by inhibitors (eg. salt, phenol, EDTA, RNA, protein…)
- Loss of the reaction during clean up
- Dead sequencing chemistry
- BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the Taq DNA polymerase or dye labeled nucleotides can have degraded
- Blocked capillary
Solving DNA sequencing reaction failures:
- Poor quality DNA:
Please using the Spectrophotometer to check DNA purity, make sure the extraction or Purification process is not contaminated by some inhibitors eg, salt, phenol, EDTA,RNA and protein, because those material will kill the sequencing reaction. Even you elute the DNA with incorrect water ( eg. Aicd pH, water with salt, water is contaminated with other nucleic acid, nuclease...) it will affect the sequencing results.
- Degraded or failed synthesis primer:
Don't use old diluted primer stocks. Don't use other peoples stocks. If you have any doubt about how the primer quality through it out and make up a fresh working solution from the primer stock. If you suspect that the primer is poor quality either have it presbyters or check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems.
- Too much template DNA:
This can be avoid by checking the concentration of the template on an agarose gel before sequencing. This will also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration.
- Wrong primer used:
Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.
- Bad water:
Inhibitors (low Ph water, nuclease...) can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap.
- DNA template is contaminated with inhibitor ( eg.salt, phenol, EDTA, RNA, protein…):
Make sure the extraction or purification process is not contaminated by some inhibitor eg, salt, phenol, EDTA,RNA and protein, because those material will kill the sequencing reaction. Even you elute the DNA with incorrect water ( eg. Aicd pH, water with salt, water is contaminated with other nucleic acid, nuclease...) it will affect the sequencing results.
- Loss of the reaction during clean up:
After sequencing reaction, we'll perform the ethanol precipitation protocol to clean up the sequencing reaction, There are a number of kits that work very well, unfortunately they can be very expensive. One tip for avoiding loss of the reaction DNA pellet when using the ethanol protocol is to add 1?l of a 20 mg/ml solution of glycogen (Sigma G-1508) to the sequencing reaction before adding the ethanol. This helps make the pellet visible and the glycogen does not seem to interfere with the injection of the sequencing fragments onto the sequencers capillaries.
- Dead sequencing chemistry:
This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles.
- Blocked capillary:
We'll clean our capillary systems every week in our lab, remove the bubbles as possible, we also change the filter periodically to avoid capillaries to be blocked.
- Why does the newly prepared primer work but fail later?
- Low pH condition and contaminated with DNase may cause primer degradation.
- Mixing after thawing was insufficient for precise concentration of primer.
- Primer with repeated freezing thawing may be damaged.