8 replies to this topic

[Miba85]

Hi everyone,

I'm currently doing research which involves multiplex PCR. My mentor tells me to freeze my DNA samples after I isolated them.
She says that improves PCR results, but she can't explain me why. A search on google scholar didn't help me, so do any of you have an idea?

Thanks in advance!

Miba85

EDIT: The DNA samples are collected from colonies on MacConkey plates, suspended in 100 µl of destilled water. (which should be enough to lyse the cells, right?)

Edited by Miba85, 13 May 2011 - 01:56 AM.

[Trof]

As I understand it, you don't really isolate DNA, you just lyse the cells and then do PCR? In that case freezing may somehow help the lysis.
Anyway using dH2O without proteinase make your DNA in lysate unstable, nucleases will be still active.

[Nephrite]

Hello :-)

I also have a question about temperatures, cDNA and RNA.

Today I heard that these samples can easily survive at 4 deg.C for a few days up to a week, even the RNA - is that true?

[gebirgsziege]

Suspending the cells will not lyse them, the lysis of the cells happens in two steps during the protocol you described: (i) by freezing your samples you create ice crystals in the bacterial cells which will destroy some of the cells (i.e. ice has a larger volume than water). This facilitates the lysis of the cells prior to (ii) cracking them open by "cooking" them in the PCR.

If you plan to use your samples more than one/two times you should think about something to stabilise the DNA as suggested in the previous post.

[gebirgsziege]

Nephrit, on 13 May 2011 - 04:02 AM, said:

Hello :-)

I also have a question about temperatures, cDNA and RNA.

Today I heard that these samples can easily survive at 4 deg.C for a few days up to a week, even the RNA - is that true?

depends on the strain of bacteria that you are working with....there are many ecotypes that have a psycrophilic lifestyle and can actively live at low temperatures such as 4°C - so genetic material found in these cells will definitly be active - although at different levels than at ambient temperature (probably a completely different set of genes). If your question is targeting towards storing your samples at 4°C for some time before processing them - this will not be possible because if your bacteria survive the RNA profile will very likely be completely different after storage.

[Miba85]

Trof, on 13 May 2011 - 02:16 AM, said:

As I understand it, you don't really isolate DNA, you just lyse the cells and then do PCR?.

Yes, that's the way it's done. About 5 colonies are taken off the plates and brought into dH2O. There is no purification prior to the PCR. I was too quick in my explanation above.

gebirgsziege, on 13 May 2011 - 04:04 AM, said:

Suspending the cells will not lyse them, the lysis of the cells happens in two steps during the protocol you described: (i) by freezing your samples you create ice crystals in the bacterial cells which will destroy some of the cells (i.e. ice has a larger volume than water). This facilitates the lysis of the cells prior to (ii) cracking them open by "cooking" them in the PCR.

If you plan to use your samples more than one/two times you should think about something to stabilise the DNA as suggested in the previous post.

Okay, thank you for your answer! I will implement that in my study report.


Thanks for all the quick replies

Miba85

[Nephrite]

gebirgsziege, on 13 May 2011 - 04:11 AM, said:

Nephrit, on 13 May 2011 - 04:02 AM, said:

Hello :-)

I also have a question about temperatures, cDNA and RNA.

Today I heard that these samples can easily survive at 4 deg.C for a few days up to a week, even the RNA - is that true?

depends on the strain of bacteria that you are working with....there are many ecotypes that have a psycrophilic lifestyle and can actively live at low temperatures such as 4°C - so genetic material found in these cells will definitly be active - although at different levels than at ambient temperature (probably a completely different set of genes). If your question is targeting towards storing your samples at 4°C for some time before processing them - this will not be possible because if your bacteria survive the RNA profile will very likely be completely different after storage.

Thank you very much for your answer, but my question was about storage of already isolated RNA and already generated cDNA from any kind of sourse material(I particularly work with archive histological materials) - I was surprised to hear today that cDNA and RNA can survive a few days in fridge.
Anyway, it was very educational to read your posts.

[Dutch]

There is a lot of data that suggests freeze thaw, and especially the conditions under which you perform them, can be detrimental to certain proteins.  However DNA tends to be fairly robust to freeze thaw.  So what may be happening is that by freezing and thawing is destroying loose proteins and proteases and effectively purifying your DNA solution.

The question is while this gives "better" PCR results, is it actually a best practice.  There are surprisingly many publications on freeze thaw in general.  Ultimately the best answer to all of your questions about DNA, RNA, and cDNA storage is to do a thaw study and confirm what works best in your process.

I do PCR as well.  We were getting concentration shifts in bulk oligos.  Turns out inconsistent thawing was causing heterogeneity within tubes.   When we would aliquot from the tube, the concentration of the tube would then drift.  Furthermore, surface tension in the narrow Matrix tubes we stored them in, made it so that even vortexing was not a sufficient remedy.  We performed similar studies, found out some things we didn't anticipate, then developed optimized thaw procedures based on that data.  It has yielded better process control data (I'm so pumped about it, I'm raising this point in the many threads on the topic).

FYI, we thaw almost everything on Box Scientific thaw stations now.  They use ambient air convection to speed up thaw times at room temperature.  This way we don't risk heat damage of cDNA and RNA.  Also, its a fixed platform like a heat block, so once we found an optimum thaw time we proceduralized it, hence our better reproducibility.

Hope that helps.

[Trof]

Nephrit, on 13 May 2011 - 04:34 AM, said:

Thank you very much for your answer, but my question was about storage of already isolated RNA and already generated cDNA from any kind of sourse material(I particularly work with archive histological materials) - I was surprised to hear today that cDNA and RNA can survive a few days in fridge.
Anyway, it was very educational to read your posts.

Once I forgot cDNA on ice and that ice melted and samples were floating in the water next morning. We re-run the real-time PCR and samples give consistent results, just the Ct was one cycle higher.

Also once we had mix prepared for run and our cycler broke down, it took a week until they fixed it, and the samples were waiting in the fridge all the time and went fine.

So, I think it can survive pretty much