cDNA Dilution step

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cDNA Dilution step

Postby marcpaulo » Mar 15 2017 9:03 am

With some kits, you make up 20ul of cDNA, and you can store this cDNA at -20oC.

Then in the PCR step, you are asked to dilute 5ul of this cDNA by 80X but are recommended to only do this immediately before you start preparing the PCR plate, does anyone know any biological or chemical reason why?

Is it due to the high dilution factor, or is it the company just trying to prevent you from making up significant reusable copies of cDNA?

If the dilution factor is too high could you do the same step with a lower dilution factor, i.e.
5ul of cDNA diluted at 4X and stored in several Eppendorf's at -20, then diluted by 20X before you start preparing the plate?

Any help with this would be great.
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Re: cDNA Dilution step

Postby relaxin » Mar 16 2017 3:48 pm

The 1:80 dilution is too high for storage. Some molecules may get lost by binding on the wall of the tube.

Your two-step dilution should be fine. The 1:4 dilution can be store frozen. Just remember to let the solution thaw complete and mix well before pipetting.
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