questions - (Dec/18/2005 )
i need your help please to answer my questions...........
In my project I use lyophilized tissue that stored at -20 C for 6 or more months, is this will be different in quality and quantity than fresh one? Why?
Why I need the replica of one species samples in each protocol (I have 3 different species extracted by different protocols for each species use replica (5 samples) so, what the important for these replica ?
When I quantify DNA for succulent plant by flurometer it gave high ratio from 1.9 and purity above 110%, can I do something to minimize reading (like add water to cuvatte till I get best result) or just take this reading? And why this plant gets high ratio and purity (only because this plant contains water)?
To ans the first question - yes it will be different in quality -- enzymes in tissues do act on their substrates and denature and change over time, even if they have been frozen at -20. Thus, they would alter the nature of the tissue. Fresh tissue also has active enzymes that will make it behave differently. However, the difference might be small enough to be ignored for the purpose of your project depending on what exactly you want to do to the tissues - for eg..if its DNA that u need to extract it will make NO difference.
replicates (i am assuming thats what you mean by replica) are ESSENTIAL in all experiments, it proves that your results are reproducible in the same conditons over and over again - and that yr experiment actually works and was not a fluke!
water has nothing to do with the ratio - you could try diluting the DNA sample, but this does not necessarily account for the high ratio. You should check that the flurometer you are using is sensitive enough to pick up the amount of DNA you have. I would also re-try the extraction with a longer digestion step.
All the best!!