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How to check if my primers were degraded?

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#1 Nephrite



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Posted 11 August 2017 - 09:14 AM


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.

Thank you!

Edited by Nephrite, 11 August 2017 - 09:16 AM.

#2 labtastic



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Posted 14 August 2017 - 03:57 PM

I assume all other components of your PCR mix are good? As in, you've run positive control amplifications with your polyermase, buffer, dNTP's, template, etc and specifically narrowed down the issue to primers?

In which case, it will require less time and money to just buy new primers than to diagnose the issue. Primers are 5-15$ each and can arrive in two days or even 1 day if you pay a little more for rush delivery.

Edited by labtastic, 14 August 2017 - 03:57 PM.

#3 Nephrite



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Posted 28 August 2017 - 02:32 AM

Thank you for your repl, labtastic.

I froze for now this task, I will reopen it in near future and I will check all the ragents.

I am concerned about the primers because I have 28 seq. for which I paid 350 euros - I can`t affor this expence anymore.

Because of my very easy luck in the past, I made my current task too ambicious - I didn`t expect such a failure.

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