Hi,
I work with bees and I have to amplify a fragment (the region from the tRNAleu - Cytocrome oxidase II, approximately 1000bp) in mtDNA. Unfortunately, the reaction does not have any amplified.
In what is wrong?
-Gene= [tRNA leu---------intergenic------COII]
-Annealing Temperature is right (calculated on the basis of primer sequence)
-Kit used= Euroclone
-DNA extraction from bee legs with Chelex 5% method (100ul of Chelex 5% + 20ul of Proteinase K. Then a termic cycle.)
-Contained in Dna very high (about 200-300 ng/ul), but impure.
How I can purify it?
Any suggestions?
For more information ask me...
Thanks
Mario
Ps- sorry for my english.
Maybe you should use phenol:chloroform:isoamyl alcohol (25:24:1) to purify your sample, then try to amplify! Or you should try some other primers to check if the problem is in your sample or in the primers. You should also lower the temperature of annealing to check ;) Hope these will help you
selecting annealing temperature by calculation only is risky, since you never know if calculation salt parameters fit to your purification method. Starting at lower temperature is a good idea. Purification of DNA could indeed be important since you never know what inhibiting substances are left from the bee legs ... would try some purification kits (Qiagen ...). Much success with your PCR
increased MgCl2 conc, TritonX100, and as given by Roland Kirchner, lower temperature will also give some amplification....
remove PCR inhibitors, washing your DNA sample with 100% ethanol or isopropanol, or dilute more your sample.
- Badges
- Science method