With the development of all kinds of research, sequencing gradually plays a more important role in the lab.Here I list several common questions that will be needs in the process of sequencing.
Q: What are necessary for sequencing primers?
A: The specificity can be combined with template. The mismatch can not be more than for basic groups. It can not contain mix-bases and the length of bases should be above 17 and below 25. It should have high purity and not be dissolved by TE buffer.
Q: What kind of solution is better for DNA sample dissolution?
A: It’s better to choose sterilized distilled water for dissolution. The sequencing reaction of DNA is also the polyreaction of Taq polymerase, which needs a most suitable condition of enzyme reaction. Someone may choose the TE buffer for the dissolution. Indeed, if we choose the buffer, the stability will be increased during the the period of preservation. However, it will lower the polymer properties after sequencing.
Q: What kind of DNA sample form is better?
A: We recommend thallus. It can remain the stability of the sample. If you really want to provide a DNA sample, pay more attention to the quantity and purity of sample, especially PCR products.
Q: If I choose the thallus, what form of thatllus is better?
A: The common forms include plate culture, stab culture, glycerol extender and fresh bacterium solution. It’s better to choose stab culture or fresh bacterium. It’s really inconvenient for the transportation of plate culture. The petri dish is fragile and the customer needs to send the sample again. The glycerol extender is more easily polluted than other methods, so it is also not a good option.
Q: How to preserve synthesized primers?
A: Undissolved primer is stable enough to preserve for at least one year with the temperature of -20?. And dissolved primers can be preserved after attenuation.
Q: Why my PCR primer can not be used for sequencing?
A: Not all kinds of PCR primers are suitable for sequencing. Here are inappropriate types:
Degenerate primer. Degenerate primer has several binding sites, which will influence the result of sequencing.
Random primer, such as the RAPD Primer. It can not fit perfectly with the template.
Overlong primer. The purity can not be guaranteed if the primer is too long. Besides, it more easily has several binding sites under a low reaction condition.
Primer with specific marks, which is the primer with fluorescence labeling. It has interference on the basic group of sequencing. Besides, primers with a great amount of marker genes are also not suitable for sequencing. It will have effects on the mobility of DNA fragments.
Impure primer. It will make noises of sequencing.
Q: What will be needed for PCR direct sequencing?
A: Specific amplification for PCR amplification and single brand is needed. And it must be purified by gel extraction. Besides, the purity of DNA is between 1.6 and 2.0; the concentration of DNA is 50ug/ml above.
Q: How many times can I do PCR reaction by 2OD primers?
A: Generally, about 400 times.
About Author / Additional Info:
Sherry Green, from CD Genomics, http://www.cd-genomics.com , a biotechnology company which provides sequencing, genotyping, microarray service for global researchers.