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Loss of volume in PCR reaction in 96 well plate


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#1 giftfee

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Posted 20 November 2009 - 04:50 AM

hei all,

I am fighting with an Gateway PCR.The latest problem is that I pipetted 20 ul reaction last night for a Gradient PCR, using 96 well plates with that aluminum foil on top. Paranoid as I am I doublechecked that its really closed, but this morning I found less than 5 ul in each well, and of course there are no bands besides the template.

My question:
Do you make PCR reactions in those (I think Polypropylene)96 well plates and did you observe problems with that? what is different about the PCR tubes?

thanks all!

#2 Adrian K

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Posted 20 November 2009 - 08:45 AM

Evaporation of course....
:lol: even if PCR tubes is used, it will loss some volume when is not properly closed.
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#3 microgirl

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Posted 20 November 2009 - 09:07 AM

Can you use plate seals (basically clear plastic tape the size of your plate) to seal the plates better. If you do this, it is also good to just fill the outer wells with water to minimize evaporation.

#4 ShannonJ

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Posted 20 November 2009 - 12:52 PM

Like they said, the foil alone will often not give a good seal. You can place a "sponge cap" or rubber mat (with divots for each well) on top between the plate and the lid. This spacer can also help to alleviate evaporation if you choose to keep on with the foils.

BTW - you can also just clean and sterilze the mats and skip the foils as well..
sj

hei all,

I am fighting with an Gateway PCR.The latest problem is that I pipetted 20 ul reaction last night for a Gradient PCR, using 96 well plates with that aluminum foil on top. Paranoid as I am I doublechecked that its really closed, but this morning I found less than 5 ul in each well, and of course there are no bands besides the template.

My question:
Do you make PCR reactions in those (I think Polypropylene)96 well plates and did you observe problems with that? what is different about the PCR tubes?

thanks all!



#5 giftfee

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Posted 23 November 2009 - 01:52 AM

thanks, I got some PCR tyubes of a colleague, if it works now I will request my boss to buy them:)

#6 Prep!

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Posted 23 November 2009 - 03:39 AM

i ve enver heard of aluminium foils for PCR reactions... If u are talking abt a Q PCR then generally optical films are used which do notn let this evaporation thing happen!!!
and if its a normal pcr.. pcr tubes will work jsut as fine... just ensure its clicked properly before use
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#7 Minni

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Posted 24 November 2009 - 03:44 AM

what is the machine you are using?
Is there a lid temperature setting? if there is .. then set it to 104^c. the evaporation problem will not be there.
Or else you can try using minreal oil.

hei all,

I am fighting with an Gateway PCR.The latest problem is that I pipetted 20 ul reaction last night for a Gradient PCR, using 96 well plates with that aluminum foil on top. Paranoid as I am I doublechecked that its really closed, but this morning I found less than 5 ul in each well, and of course there are no bands besides the template.

My question:
Do you make PCR reactions in those (I think Polypropylene)96 well plates and did you observe problems with that? what is different about the PCR tubes?

thanks all!



#8 warsel

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Posted 25 November 2009 - 05:17 AM

I usually use biorad's PCR plate seals and those work fine - I never have evaporation from 96-well pcr plates since I switched to these (you need a seal-roller though).




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