Hello,

I'm having some difficulty with PCR inhibition. I'm past the point of optimizing my PCR reaction, and I am currently screening my organisms of interest for the presence of prey DNA in their guts. During my optimization I wasn't having any issues with inhibitors because I was diluting my stock DNA solutions from ~400ug/ul to 25ug/ul. I can not dilute my samples without risking false negative results. I am running 2 different multiplex reactions, one that screens for 3 prey species and another that screens for 2 prey species. My reaction looks like..

(3 prey multiplex)
27.8ul H20
10ul buffer
6ul primers (1ul of each primer for 3 species forward/reverse)
1ul of predator DNA (may or may not contain prey DNA)
0.2ul Taq Polymerase
5ul dntps
--------
50 ul Total

I am extracting my DNA using DNeasy blood and tissue kits. My predators/prey are phloem feeding insects and there are polysaccharides and other nasty substances in their guts that can cause inhibition. I've tried a few post DNA extraction inhibitor removal procedures and so far PVPP columns seem to work best but I'm still getting iffy results.

Now getting to my question. I'd like to try adding BSA to see if that helps at all but what should I try? like 1ul (10mg/ml) BSA solution for example or is that too little?

If anyone has any suggestions regarding BSA or any other suggestions regarding other inhibitor removal procedures or any other suggestion that may help me get consistent results I would greatly appreciate it.

Thanks Aga, Actually the degenerate primers were designed by multiple sequence alignment of protein sequences of target gene in different bacteria. The bacterial strain on which I am working, nothing is known about the genetics of the target gene. There is only one report that too is not exact gene but related and in that paper also they didn't find any matching sequence in the nucleotide database and after converting to protein sequence they found 57% identity with the target gene they were studing in the same bacterial strain I am studing.

These primers have been tested on different bacterial strains and they amplify the target gene. I was wondering if the nucleotide sequence of the target gene is unique in this organism. How should I justify this from publication point of view. Waiting for expert suggestions.

Hi,

I just want to know if anyone adds beta-mercaptoethanol to their 10x PCR buffer and if so how much 10 mM beta-mercaptoethanol would need to be added to say 100ul of 10x PCR buffer?

Thanks in advance.

Hello!
Did anyone work with gadd45 and its mRNA expression?
I wonder about the high or low abundance of gadd45 in normal cells?
I know that 28s or 18s as reference genes have high abundance in the cells.

Thank you,
Kobrus.

splicing of specific mRNA? I know the very basics of how RT-PCR works so I am not really sure how this would relate to alternatvie splicing. Any information would be helpful.