PCR primer resuspend method? - (May/17/2007 )

I want to learn the way to resuspended oligos from lyophilized (dry form)?. Can anyone tell your experiences on it?.

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I just use Tris-EDTA (10mM Tris 1mM EDTA) to resuspend, then aliquot to 100ul. The amount that you use to resuspend will depend on the amount of the primer. The amount of each primer will usually vary but the paper work you got from your vendor should list it somewhere. I try to resuspend to a concentration of 20pmol/ul then use 1ul of each primer for a 25ul reaction mix.

Jeff

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YOu can either use TE buffer (usually provided) or just normal water will do.

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I just add water to the lyophilised primer for a final primer concentration of 0.4nmol/ul for my stock solution, then take 1ul of this to make a 10pmol/ul working solution (1:40 dilution). I usually use 0.5ul of this working solution for a 20ul reaction volume.

It is always a good idea to have a stock and then make a working solution from this, as it means all is not lost if contamination occurs

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Thanks Jeff

Could you tell in detail about calculation to get working solution of 20pmol/ul as we got 150 nmoles of lyphilized (dry form).
Thanks

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To do it as simply as possible:

20pmol/ul = .020nmol/ul

150 divided by .020 = 7500

Thus the volume of buffer added should be 7500ul

Aliquot it out into smaller amounts and keep at -20 till needed.

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how is different between nmoles and picomoles? and how many fold time different?

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The SI prefix order is:
milli
micro
nano
pico
femto

So a nanomole is 1000x a picomole.

If you have 150nmol of dry primer, resuspend it in 1.5mL of TE. This will give you a concentration of 100uM, a good 'stock' solution.

Now take 10uL of this into a new tube with 40uL of TE. This gives you a 'working solution' of 20uM, which is what you want. Put the stock solution back in the freezer!

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Yes, but i also need to know the procedure to make stock solution? (eg. add TE, then centrifuge or shaking or whatever next ...). That it would be nice to know.

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Add water or TE to the lyophilised DNA, then wait for 1-2 min. the primer will dissolve instantly and then very briefly and gently vortex and store in -20C. This is your stock sol.

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