According to the Nanodrop Spectophotometer my dna sample of Holothuria tubulosa (sea cucumber) from gonads is pure.
The DNA extraction has been made with the CTAB Protocol.
The ratio is 1.91 and 2.31 for the 280/260 and 260/230. The concentration of my sample is 350 ng/ul.The curves and ratios are perfect so i think there is no problem with my samples according to nanodrop.
I have tried many (up to 35) PCR RAPD 10mer Primers with many combinations such as:
1) 2-5% DMSO
2) Different concentrations of DNA sample ( from 10-350 ng/ul )
3) Gradient PCR from 30-44C (annealing step)
4) Samples such as Holothuria tubulosa skin, Urchin and Shark (Urchin and sharks were amplified so i have positive samples.)
5) Different PCR steps such as long time denaturation (from 5 to 30 minutes), and long final extension step.
6) At 25μL and 30μL with different concentrations of Primer (0,3-3μL / 25μL), Buffer (2,5-10μL / 25μL) DNA (1-2μL / 25μL), DNTP (0,5-2,5μL/25μL) but i have no results.
Although Holothuria tubulosa gonads and skin has a problem in PCR amplification and i don't understand the reason why.
Any idea of what's preventing PCR amplification in that case?
Edited by JohnSky, 07 July 2012 - 07:09 AM.