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Primers Freeze-Thaw - (Oct/10/2012 )

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Loading ready ladders typically have bromcresol purple and xylene cyanole blue rather than orange-G as the loading dye. These blue dyes interfere with band visualization, and a far as I can tell are in every way inferior to orange-G. I've tried to convince NEB to switch, to no apparent effect. And why would I make an extra trip to the fridge if I can just keep the ladders on my bench?

-phage434-

@Mad researcher: In a water probably not, it doesn't keep pH. I was diluting everything in water while working on diploma thesis, until the moment some primers just stopped to work even though they were freezed all the time. I took advice about the Tris and dilute everything in it ever since.

@phage434: Yes they do, but our ready load mix from NEB contains only bromphenol blue and I didn't ever notice interference with ladder bands. I made my own before that, you can adjust the amount (or type) of dye.
I had more problems with the bromphenol blue loading for samples, there are many those that run around 400 bp range of bromphenol, but I adjusted the concentration and made a special "visual aid only" loading with just a bit o xylene cyanol for samples that are extremely faint in that range. Orange-G runs far too quick for our use, so it's unusable for longer runs. And what also not unimportant, we don't have it ;)

-Trof-

I would disagree with all our experts here.

Working primers diluted in water and stored at 4oC work perfectly fine for up to 2 years (that's how I long been working in this lab). Actually, even my stock primers are diluted in water but kept at -20oC

Some working primers I use on more or less a daily basis but other dont come out for months together.


and about the ladder, we keep that too on the bench, with no issues. I have gone ahead and diluted ready-made ladders with equal amounts of water and they work fine for me. So, lot of money saving and also effort saving :P

-Ameya P-

@Trof - Could you give me the recipe for 10mM tris ?

-Mad researcher-

Ameya P on Fri Oct 12 06:10:12 2012 said:


I would disagree with all our experts here.


Well that's not actually a diagreement, that's just a different experience. I had mine working for years too, then once just degraded.
Actually with the primers it's bit like with the computer disk backup, everything is so long so fine, until one day.. it's not.
It's everyones decision if he wants to risk that, but same as with backups, once you got your primes degraded/data lost, you kind of change a view on these things.

@Mad researcher:
100x diluting the 1M Tris pH8 stock with milliQ water (100 ul for 10 ml of buffer), I aliquot it into 1.5 ml tubes.

-Trof-

Do you guys suggest preparing a master mix without the TAQ and storing it at -20 for further use?

-Mad researcher-

If you store in Tris or (worse) water, you are at the mercy of accidental DNAse contamination. This is why TE is so much better. And the idea that 1 mM EDTA compromises an enzyme reaction when the amount added as primers is typically 5% of the final solution is quite absurd.

-phage434-
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