I'm tryint to standardize a PCR to mycoplasma detection of dna from cell culture. I'm testing samples positive for mycoplasma and negative as controls. I found a weak band unspecific slightly below the mycoplasma band.
Dear Bruna Rodrigues
Pls. find the attached files, may help in your research, good luck
http://www.ctcusp.org/pdf/inctc_posters/30.pdf
http://genome.cshlp.org/content/4/4/199.full.pdf
http://link.springer.com/article/10.1007/BF00127903
http://icmr.nic.in/ijmr/2013/august/0811.pdf
https://www.researchgate.net/profile/Jens_Boman/publication/11880269_Dowell_S_F_et_al_Standardizing_Chlamydia_pneumoniae_assays_recommendations_from_the_Centers_for_Disease_Control_and_Prevention_USA_and_the_Laboratory_Centre_for_Disease_Control_Canada_Clin_Infect_Dis_/links/56546f1508ae1ef92976a1a1.pdf
http://bcrj.org.br/wp-content/uploads/2015/08/Clique-aqui.pdf
https://www.researchgate.net/profile/Martin_Altwegg/publication/10875508_Development_of_a_multiplex_real-time_quantitative_PCR_assay_to_detect_Chlamydia_pneumoniae_Legionella_pneumophila_and_Mycoplasma_pneumoniae_in_respiratory_tract_secretions/links/0fcfd50bf3e45944cb000000.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949616/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463982/pdf/10616_2004_Article_5113481.pdf
Stephen Gillespie, Peter M. Hawkey. 2006. Principles and Practice of Clinical Bacteriology John Wiley & Sons,Pp. 620. ISBN 0470035323, 9780470035320
R. Ian Freshney. 2011. Culture of Animal Cells: A Manual of Basic Technique and Specialized ApplicationsWiley Online Library: Books , Pp. 768 . ISBN 0470649356, 9780470649350
Regards
Prof. Houda Kawas
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Hello Bruna,
Try the following.
1) use a gradient PCR to know the exact conditions for pro,er annealing.
2) Lot of smear is there in the PCR. Titrate the magnesium concentration.
3) Is mycoplasma GC rich or AT rich? keep this in mind for choosing the strategy.
4) Try to use Fusion DNA polymerase. This is faster and more specific them Taq.
Hope it works for you.
Cheers!
Try to standardize your annealing temperature by using gradient PCR.
Hie. You can try the LookOut Mycoplasma PCR detection kit from Sigma-Aldrich. I have been using it for the past 12 months and i don't have any regrets.
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