Plate Sealing for Real Time PCR

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Tony Rook
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Plate Sealing for Real Time PCR

Please indicate which method you prefer for sealing your real time PCR plates.

nin1318
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i have used sealing film..

i have used sealing film...but i have also found that some machines have i guess a hotter lid and it melted the seal and samples evaporated. i think that caps are the best way to go. flat caps are the easiest and probably less likely to get dents or whatever that could cause problems with the optics.

atcg
atcg's picture
We struggled so much with

We struggled so much with this issue in the lab I used to work in!! We tried a variety of brands and methods, including sealing foils, films, caps, lids, and mats. Many sealers did have different temperatures, especially around the middle, sometimes leading to evaporation on the outer wells, and melting of the plate in the middle!!! We finally settled on foils that could be "rolled" off using a small turnkey apparatus. I forget which brand and sealer we used, but I can find out if you're interested!

Tony Rook
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Jamie_Cruikshank wrote:We

Jamie_Cruikshank wrote:

We struggled so much with this issue in the lab I used to work in!! We tried a variety of brands and methods, including sealing foils, films, caps, lids, and mats. Many sealers did have different temperatures, especially around the middle, sometimes leading to evaporation on the outer wells, and melting of the plate in the middle!!! We finally settled on foils that could be "rolled" off using a small turnkey apparatus. I forget which brand and sealer we used, but I can find out if you're interested!

Jamie:

It would be very helpful to the rest of the forum if you could post the which brand and sealer you recommend. It's always helpful to hear what others have struggled to determine.

ant
ant's picture
Kinetic PCR - (now Real-time

Kinetic PCR - (now Real-time pcr) originally was performed
by Russ Higuchi under mineral oil
As it is supposed that samples will not be analysed by gel
why not to use ORIGINAL protocol with mineral oil
or "optic wax" from MJ research ?????????

Tony Rook
Tony Rook's picture
As with all scientific

As with all scientific methods improvements are anticipated and expected to further their sensitivity and/or applications. Ant, you are correct that Dr. Higuchi used mineral oil to demonstrate the applicability kinetic PCR. However, he also used a CCD camera to measure the fluorescence of each PCR cycle. Today, real time thermocyclers with integrated fluorometers has greatly improved this aspect of the original method. Likewise, plate sealing has had a variety of improvements (including optical caps, sealing film, and wax). Additionally, you are correct that when using non-specific chemistries (such as SYBR Green) a melting curve can be performed to verify amplicon purity, however, to verify purity of the PCR products when you using probe based chemistry, a gel is still required. Therefore, it is advisable to use a sealing method such as optical caps.

I am interested to know if evaporation of reaction volume is a worry when using something like mineral oil, as this will greatly vary your real time PCR results.

ant
ant's picture
As I remember last year in

As I remember last year in AnalBiochem there was a paper
about plate and volume bias in Biorad realtime

the authers conclude that edge wells have poor results not
because of EVAPORATION but because the parts of tubes-plates
that protude ABOVE the surface of block are COLDER and promote
extensive condensation!!!!!

Thut is why caps and films WILL NOT HELP

CONCLUSION - GoTo - step1(Higuchi)- MINERAL OIL-for
50 cycles - then END

Sybr green - despite melting curves - can generate artifacts

See Paper SYBR Green vs SYTO9 in realtime

-

ant
ant's picture
I do not SEE how modern

I do not SEE how modern PELTIER cyclers
greatly IMPROVED the original METHOD of Higuchi

He WAS USING HIGH-performance NON-PELTIER !!!!!!1
cycler

See paper " Physical characteristics of six modern thermocyclers"

this paper describes the so-called "advantages" of Peltier
96-well cyclers

ant
ant's picture
the sealers DO NOT HAVE

the sealers DO NOT HAVE DIFFERENT temperatures
when you put the sealer onto plate especially skirted-
the plate immediatelly expands due to high coefficient of
thermal expansion of plastics and central part dome out
and comes in tight contact in the midle- this effect persists
during cycling

use non-skirted flexible plates for realtime
it will help somehow

ant
ant's picture
nice plates for realtie are

nice plates for realtie are described in user manual
for Amplifluor SNP HT Genotyping System (Chemicon)
they were tested for allele-specific PCR- this reactin
much more demanding than conventional low complex cDNAs

nin1318
nin1318's picture
Tony Rook wrote:I am

Tony Rook wrote:

I am interested to know if evaporation of reaction volume is a worry when using something like mineral oil, as this will greatly vary your real time PCR results.

mineral oil should prevent evaporation/condensation of the sample. this is its function when using a thermal cycler without a heated lid, regardless of the method of sealing your tubes. i would assume that all real-time machines have heated lids and this is only a confound with regular pcr machines.

nin1318
nin1318's picture
ant wrote: I do not SEE how

ant wrote:

I do not SEE how modern PELTIER cyclers
greatly IMPROVED the original METHOD of Higuchi

actually, it's a hell of a lot easier to just put cap strips on a plate than to pipet oil into each well. minimizing the time of sample preparation during pcr prep is always beneficial.

nin1318
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sorry i should have said heck

sorry i should have said heck not h e l l ahahaha

ant
ant's picture
Analytical biochemistry 337

Analytical biochemistry 337 ( 2005) pp347-350

heated lid cannot enshure UNIFORM condensation
in ALL wells of 96 cycler thus changing the TEMPERATURE
of SAMPLES with volumes bellow 50 mkl

The MAIN QUESTION of TOPIC

must be corrected

What SEALING option is GOOD for

10 mkl volume
20 mkl volume
30 mkl volume
40 mkl volume

........
150 mkl volume

????? :)

ant
ant's picture
uniform caping of 96 plates

uniform caping of 96 plates is not much more faster and
reliable than oil-caping of 384 plates ????
Who is the FIRST HERO ????

ant
ant's picture
www.kbioscience.co.uk

www.kbioscience.co.uk

have a LASER PLATE SEALER

may be it is le best option ??

Have not tested it but somebody can try

ant
ant's picture
finally VOTING has NO

finally VOTING has NO practical sece untill

1. Cycler - company- model ????
2. Plate (48- 96- 384-)- company - cat N
( a lot of plates cannot be used even for standard PCR)
3. Volume of reaction

ONLY AFTER - SEALING OPTION

nin1318
nin1318's picture
ant wrote:uniform caping of

ant wrote:

uniform caping of 96 plates is not much more faster and
reliable than oil-caping of 384 plates ????
Who is the FIRST HERO ????

what are you talking about?