vanu

Hi all,

I need to prepare sodium acetate buffer 3M at pH=5, I have sodium acetate tridydrate (PM 136,08 g), can I prepare it from this one?
I have tried to prepare 50 ml, but find quite difficult to dissolve it and also difficult to adjust the pH with HCl 37%, could someone help me please?

Thank you very much

Dear all,


I would like to ask about a problem that has come to me when analyzing the agarose gels I have performed with my PCR products.

I´m trying to find the optimum annealing temperature for my pair of primers, therefore I have conducted a PCR reaction for each of  my pair of primers and have visualized them in an agarose gel.

The problem is that the lanes I obtain in the gel, even if they are strong and clear, are smaller than the lanes obtained from the DNA Molecular Weight Marker that I use (DNA Molecular Weight Marker X 0.07-12.2kbp). Therefore,  I´m not sure if the lanes I´m looking for in the gel are the result of primer dimers or in contrast, they are the result of a specific PCR reaction, where the obtained DNA size is small.

This image is an example of the lanes obtained in one of the runned gels.



Whereas, in the next figure (other pair of primers that analyze the same sample) two aspecific lines of lanes could be observed.


I would really appreciate if someone could help and give me any advice in relation to this, as these PCR products that I want to obtain will be to send for next generation sequencing once purified.

Thank you very much in advance for your help,

Dear all,

I would like to ask whether nobody has had the similar problem I´ve had with the DNA I extracted from rats stool samples.
The problem is that these animals have been supplemented with phenolic compounds and when the DNA is extracted from their stool samples even if the ratio 260/280 is admissible (aprox. 1.8-1.9) the 260/230 is too low (0.7-0.9) (I suppose it could be because of the phenolic contamination), when compared to the ratios that I obtain from the samples which have not been supplemented.

In addition, when I performed the PCR with these samples, the results are worst than the results I get with the samples obtained from rats that have not been supplemented.

For the DNA extraction I use QIAamp DNA Stool Mini Kit, so my question is whether you could advice me about any possible alternative/solution, or if someone has had the same problem, the way he/she solved it....


Thank you very much in advance for your help,