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way to know if my UNOII pcr machine is working properlly


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3 replies to this topic

#1 unoII

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Posted 17 July 2009 - 11:11 PM

hi

i am working now for two months doing pcr and doesnt manage to get reliable results, It seems that if i am using pcr of another lab i get a band but if i am using my ocr machine i don't. is there a way to self check my UNOII biometra pcr machine?


ben

#2 HomeBrew

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Posted 18 July 2009 - 06:15 AM

Hi unoII -- welcome to the BioForums!

Seems like you may have already done what I would suggest first -- make a complete PCR reaction mix (primers, template, dNTPs, MgCL2, etc.) that is twice the volume you'd normally use. Split this mixture equally amongst two tubes. Run one tube in your machine, and the other the machine from the other lab, using the exact same PCR program in both machines.

If you get an amplicon from the other lab's machine, but not from yours, there's likely something wrong with your machine.

In your machine, set a program for the block to cycle between something like 4C and 37C, 10 seconds each loop. Run the program and feel the block with your hand -- is the temperature changing?

Do you have a manual for your machine?

#3 ivanbio

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Posted 20 July 2009 - 08:16 AM

I had this happen to me a while back when I was running a PCR reaction in an Erincomp (sp?) and an MJ. Bottom line the reaction worked on the MJ and never did on the Erincomp. Everything else equal I agree that if the reaction does not work on one machine and it does on another, there is something wrong with the machine. Yet, you may also want to consider that each PCR machine works differently (different ramp rates, etc), so it is possible for a PCR reaction to not work on one machine and work on another (assuming the PCR reaction is not very robust and just barely working).

My two cents

Ivan
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#4 HomeBrew

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Posted 20 July 2009 - 01:44 PM

...you may also want to consider that each PCR machine works differently (different ramp rates, etc), so it is possible for a PCR reaction to not work on one machine and work on another (assuming the PCR reaction is not very robust and just barely working).


A good point -- if your PCR is marginal, perhaps selecting new primers would help?




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