I try to reproduce an experiment that I did abroad some years ago. It was a preliminary research and everything worked since the beginning as it should be. No problems, no troubleshootings - just fine specific bands and single melting curves. All results were reproducible.
Here and now I have a real catastrophy. Nothing works. I have awful smears, non-specific amplification or nothing.
Of course, I try to work as clean as always, but.......
I suspect nuclease contamination and I need to check if at least my primers are safe - unfortunately they are expensive and I prefer not simply to throw them out.
Here are my questions:
1. The primers are 18-23 nucleotides. What is better - resolution on 4% agarose or 20% PAGE and why? Because of the percentage of the gel?
2. In the case of 20% PAGE, I guess I use the vertical electrophoresis equipment, right?
3. I guess I should cast also the 4% stacking gel, right?
4. What buffer I use - TAE?
5. Which of the solutions I load - the stock of 100 pmol/ul or the working of 10 pmol/ul?
6. I guess I still use 6x DNA loading buffer, correct?
7. I guess use of ultra low DNA Ladder is imperative, right?
Any help or advise will be highly appreciated.
Edited by Nephrite, 11 August 2017 - 09:16 AM.