I work on Torque Teno virus (TTV) with Takahashi primers
‘T801’: 5-GCTACGTCACTAACCACGTG-3 (sense, nt 6–25),
‘T935’: 5-CTB6CGGTGTGTAAACTCACC-3 (antisense, nt185–204;
B: mixture of G, C, and T)
The original article only provide physical condition (Takahashi, et al., 1998), while other papers used different condition with high primer concentration 50 picomole ( Kristian, et al., 2010) or low primer concentration 0.4 picomole (Bouzari & Baygloo, 2008); nothing works for me.
anyone can help me in optimization of these primers.
Thank you in advance
- First try to optimize annealing temperature by running gradient PCR from 60 °C to 53 °C using 0.5 microMolar primer concentration.
- After getting optimum Ta, try a range of primer concentration from 0.1 microMolar to 0.9 microMolar for optimum concentration(some times peoples also try different conc. combination of forward and reverse of above range).
- Importantly try to use the template conc. range from 10 ng to 100 ng.
Dear Haneen Sarairah
Pls. find the attached files
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293898/
http://jid.oxfordjournals.org/content/181/5/1796.long
http://memorias.ioc.fiocruz.br/issues/past-issues/item/2616-tt-virus-infection-in-children-and-adults-who-visited-a-general-hospital-in-the-south-of-brazil-for-routine-procedure
http://docplayer.net/3052180-Molecular-detection-of-torqueteno-virus-in-healthy-blood-donors-in-khartoum-state-sudan.html
http://geb.uni-giessen.de/geb/volltexte/2004/1467/pdf/PabstDirk-2004-03-09.pdf
https://www.ncbi.nlm.nih.gov/pubmed/15831945
https://www.ncbi.nlm.nih.gov/pubmed/17146841
http://www.scielo.cl/pdf/ijmorphol/v26n2/art20.pdf
https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-6-134
https://web.wpi.edu/Pubs/ETD/Available/etd-031509-151117/unrestricted/JGriffinMSThesis.pdf
Hoping this will be helpful
Regards
Prof. Houda Kawas
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