I am trying to clone various laccase isoforms sequences via mRNA and cDNA synthesis. I need to design a pair of degenerate primers to amplify as many sequences as possible.
Hi Brandt!
I had used PRIMACLADE. It is friendly to use and intuitive. I use to check the obtained primer sequences with mFOLD and also with OligoCalc as Russell Orr advised in answer to another collegue. If trying mFOLD, feed the application with the annealing temperature and salt concentration you are planing to use in your PCR reaction for better prediction of the secindary structure.
I am also attaching some pages extracted from a manual prepared by Qiagen (Critical factors for successful PCR) that provide guidelines for designing and using degenerate primers. Best wishes on your primer design!
Thank you so much. This information really helps. I'm going to start right now
The cDNA is from Trametes versicolor.. I am currently sequencing these isoforms. as soon as i get it done it should be possible
Thanks.
What do these laccases synthesize/brake down? I know that fungi can use it for many functions, cell wall brake down, cell wall synthesis, melanin production.
The phenotype I am looking for is melanin production. Do your isoforms produce melanin?
No problem,
In my case I´m intersted in cloning and expressing these isoformsfor waste water treatment. Melanin synthesis by laccases is more common in bacteria. How ever I do believe that the isoforms that my fungus produce are capebale of producing melanin.
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