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I'm planning to order oligo, polymerase, nucleotide, buffer for my first diy PCR experiment (now I have nothing), so I have some questions.

  1. How and how long I can storage them? I have only a fridge that can freeze to -10 -> -20C.

  2. I see the size/concentration of polymerase is 400 units and 25 nmole of oligo. So what can I do with 25 nmole oligo and 400 units of polymerase? How many PCR experiment I can do with 400 units of polymerase, and how many PCR experiment I can do with 25 nmole of oligo?

  3. If I have a HPLC machine, can I reuse the polymerase by purify it after reaction?

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  • $\begingroup$ At -20 C, you can keep things around for a pretty long time. I'm not sure that years is a good idea, but at least months. $\endgroup$ – blep Jul 3 '13 at 0:47
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The manufacturers of each component should have information available on their website regarding storage temperatures, along with recommended protocols on how much of each component to add to the PCR reaction. I would not try to reuse the polymerase, as depending on the kind you buy it may be a complex of proteins, and it may not be functional after purification. However, you're welcome to experiment all you want, it's your stuff!

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  • 1: -20 is the normal storage condition for PCR reagents in the lab so you should be ok. One warning: you should make sure that your freezer is NOT auto-defrosting. Most consumer freezers will cycle temperature to prevent frost build up. This is very bad for enzymes and will greatly reduce their lifespan. If you can’t find a non-defrosting freezer, you might be ok if you buffer the temperature by keeping your enzyme in a cooler in the freezer, or inside a block of metal with a hold drilled in it.

  • 2: You can stretch your reagents by lowering the volume of each PCR reaction. 25uL is workable in the lab, but keep in mind that the smaller the volume the larger the relative error in your measurements becomes.

  • 3: It seems strange to me that you have access to a HPLC machine but can’t afford to buy new enzyme when you run out. I really wouldn’t try to reuse anything. Maybe it could work, but would you really trust your results? If you shop around you can find cheaper brands of polymerase.

Good luck!

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  • $\begingroup$ 3: depends on the polymerase: some of the more expensive polymerases will loose some of their functionality (e.g.: "breaks" that avoid accidental polymerization prior initial heating) $\endgroup$ – tsttst Oct 26 '17 at 0:25

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