Wilsea Maria Figueiredo

• Volunteer at Laboratorio Monitora

A need to analyse specific sequences gathered from genbank in order to analyse primers matching/designing. I have used Sequencher (gene codes) in the past and I loved it. Yet prices are now prohibitive. Any suggestions?

Having problems with dimers and an answer from Paul Rutland for another question made me think that the fact that I store my PCR mix in the fridge might be the problem. I must add that in this mix I add both primers, so maybe dimers are being formed prior to the reaction during storage. Does this make sense?

I have so far disliked Taqs that come with dyes because dye concentration seems excessive to me. I have recently bought a Taq with dye-free buffer, I wonder if I can use a small bit of gel loading buffer inside my pcr reactions. Thanks!

Hi all. I have some primer pairs which always produce those horrible primer dimer bright smear! Increasing annealing temperature does not solve the problem. Any suggestiopn for a PCR enhancer or another strategy? So far I have used DTT and DMSO and amplification quality still poor! Thanks

Need do look for milk bacterial pathogens using PCR. Does anyone suggest me a good protocol for DNA extraction? I have heard milk proteins are difficult to deal with aabd act as taq inhibitors

I am making tests for diagnostic PCRs. At the moment I am trying to standardize a pcr reaction for a circa 180 bp product. I did not get positive bands so far. Interestingly, I get what appears to be a primer smear in both negative and positive controls. But no smear at all for DNA samples I do not know abou pathogen absence/presence. Any idea why this is happenning?

We would like to preserve RNA from mammal blood samples. Unfortunately the only product I am aware is RNA Later and it is very very expensive for our limited budget. Any ideas? Thank you very much

Sorry basic question. I am aware primers should be described in the 5'-3' direction in the literature. When I am ordering primers I have taken from published papers, should I use the same sequence or make a reversed and complemente strand for ordering the reverse primer?

Hi all, does any of you know of specific primers for both emetic and diarrhaeic forms of Bacillus cereus?

We are currently having problems with PCR amplifations of a ~200bp gene fragment. We cannot see any product after a 35 cycles amplification round and usually use a simple reamplification protocol: 1ul of our first PCR to a 20ul reaction. Resulting positive bands are not so clear so I wonder if you could kindly share your experiences with reamplification protocols. Thank you!