9 replies to this topic

[nightingale]

opening the cycler today morning, to bring out my first round PCR tubes,
realised there are water droplets on the heat-block ...
exactly on the edge-wells ...

so i was " ????? "
is it condensed water ??? But, if so ... what is the source ???

grateful to any insights ...

p.s : is this a sign that the cycler is getting out of date ?

[phage434]

If this was a typical pcr protocol, it held the samples after cycling at 4C indefinitely.  That was probably 10-12 hours.  With any leakage of air into the sample holder, condensation of humidity will occur on the block.  This is not surprising, nor worrying, but expected.

[nightingale]

phage434, on 26 April 2011 - 11:48 AM, said:

If this was a typical pcr protocol, it held the samples after cycling at 4C indefinitely.  That was probably 10-12 hours.  With any leakage of air into the sample holder, condensation of humidity will occur on the block.  This is not surprising, nor worrying, but expected.

thank you
this was the first time i see such thing, so i was worried :S
thanks ...

[neuropath]

nightingale, on 26 April 2011 - 11:57 AM, said:

phage434, on 26 April 2011 - 11:48 AM, said:

If this was a typical pcr protocol, it held the samples after cycling at 4C indefinitely.  That was probably 10-12 hours.  With any leakage of air into the sample holder, condensation of humidity will occur on the block.  This is not surprising, nor worrying, but expected.

thank you
this was the first time i see such thing, so i was worried :S
thanks ...

Some manufacturers recommend holding at 10C. Less condensation. If the condensation seeps into the circuit boards, you'll have a dead machine. 10C is just as good as 4C for preserving your samples for short periods

[Trof]

neuropath, on 01 May 2011 - 07:19 AM, said:

Some manufacturers recommend holding at 10C. Less condensation. If the condensation seeps into the circuit boards, you'll have a dead machine. 10C is just as good as 4C for preserving your samples for short periods

I think the main reason for this recomendation is to prolong the lifetime of Peltier blocks, which get exhausted by maintaining low temperatures for longer time.

[nightingale]

thanks alot for the valuable info. given
i will keep an eye on the cycler.

neuropath: & what does "short period" means, overnight for example ???

[neuropath]

nightingale, on 02 May 2011 - 11:35 AM, said:

thanks alot for the valuable info. given
i will keep an eye on the cycler.

neuropath: & what does "short period" means, overnight for example ???

The longest I've tried is overnight around 12 hrs. I hardly need to do that these days after I discovered Kapa Biosystem's fast enzymes. At 1kb extension per sec (yes, sec!), your typical run is over in less than 30 min. I use their slower enzyme (cheaper) but its still 2kb/min. As for pricing, its comparable with other brands

P/S I do not work for Kapa or own their shares!

Edited by neuropath, 04 May 2011 - 05:46 AM.

[nightingale]

Quote

The longest I've tried is overnight around 12 hrs. I hardly need to do that these days after I discovered Kapa Biosystem's fast enzymes. At 1kb extension per sec (yes, sec!), your typical run is over in less than 30 min. I use their slower enzyme (cheaper) but its still 2kb/min. As for pricing, its comparable with other brands

P/S I do not work for Kapa or own their shares!

per second ! ... that's really FAST !!!

p.s : are u sure of that ???!!!

[Adrian K]

I read the protocol, it says:

1 sec at 72 °C for amplicons ≤1 kb, 15 sec/kb at 72 °C for amplicons >1 kb

but then, the website also mention: Fragments up to 5 kb may be amplified from plasmid or lambda DNA with KAPA2G Fast, but fast amplification of genomic targets >3.5 kb is not recommended. (http://www.kapabiosy...g-fast-pcr-kits)

@neuropath: I wonder how long is your fragment that you need 12 hours to do your PCR last time.

[neuropath]

adrian kohsf, on 04 May 2011 - 09:43 PM, said:

I read the protocol, it says:

1 sec at 72 °C for amplicons ≤1 kb, 15 sec/kb at 72 °C for amplicons >1 kb

but then, the website also mention: Fragments up to 5 kb may be amplified from plasmid or lambda DNA with KAPA2G Fast, but fast amplification of genomic targets >3.5 kb is not recommended. (http://www.kapabiosy...g-fast-pcr-kits)

@neuropath: I wonder how long is your fragment that you need 12 hours to do your PCR last time.

It wasn't the reaction time. It was the cold soak at the end of the run. That's how this topic started