Would it be possible to aliquot out your cDNA and store at -20oC and take one batch each time to avoid freeze-thaw and prevent any worry about storage at 4oC? I have kept my cDNA in the fridge before without any problem but never more than 2-3 days just because of the way my experiments worked out.

Hi, I agree with Sarah: make some aliquots (don't forget to ix your cDNA sample before you make aliquots) and then store it at -20°C. That's a save way and a way to avoid contamination (if you are using the cDNA form one tube without making aliquots). Cheers, Nadine

-20 degrees

-20 degrees

I would store cDNA at -20°C, but if you are going to do many PCR with the same cDNA in different days, I would prepare different cDNA aliquots

-20°C. Avoid repeated freeze thawing

I always use aliquots, because after 5-6 freeze-thawing, I observed lose of activity.

-20°C would be better, I agree with Jacopo for aliquots if you will do many PCR in the short future.

We always freeze cDNA at -20ºC

-20ºC will be best. I will make aliquots, as is previously suggested, according to your needs, for example have one aliquot for your every day PCR, so you do not need to thaw and unthaw.

Repeating the same answer for getting more RG points?

Its better to store cDNA aliquots at -20ºC for single use to avoid any DNase contamination while repeated pipetting and also to avoid repeated rounds of freezing and thawing.

-20°C; stable for at least 3 m. Avoid repeated freeze thawing although can do it couple of times. Alternatively - aliquote the prepared cDNA.

As Nadine suggested repeating the same answer does not make to much sense...

I have different suggestion: if you repeat the PCRs within one week, I would leave the cDNAs at 4°C. We have done RT-qPCRs using cDNA stored at 4°C and have not seen any differences for the control gene within one week.

I've had great success storing cDNA at 4C for a week at a time -- reagents too. The freeze thaws do more damage and throw off the concentration far more than storing in the fridge...

I have stored my cDNA at 4 degrees for 3 weeks and it worked all the time.

Its better to not store much time and avoid repeated freeze thaws which led breaking of terminal (3`OH) group and inactivation of c DNA for further work. Another thing what you can do make many aliquots and store at -20C for week/ two weeks and use one time after freeze thaw exhibit better results.

I keep my cDNA aliquots at -80C to maintain stability. After I generate I aliquot to avoid multiple freeze/cycles. However, I make large batches of the same cDNA and store for years, and use to subclone ORFs from a defined fly genotype. I can see that this becomes impractical when you have a lot of samples. However, I would not store at 4C due to the higher instability compared to plasmid and genomic DNA. In my case, I store all my DNA (plasmid, genomic, primers) at -20C, except for cDNA and DNA that is used for microinjections. The single exception are extreme sized BACs that I will sometimes keep at 4C for short times.

aliquot in -20C. or 4 degree for a few weeks should be fine.

if would keep one aliquot at -20º (just for security) and one other at 4º that I would use for the experiments during that week (I have stored DNA at 4ºC for up to 1 month with good results)

Yes, one must always store cDNA in -20 degree Celsius to avoid its degradation. Being single stranded, cDNA is more unstable than the double stranded DNA, therefore, more care is required during storage.

As others have suggested it is a good practice to minimize degradation by storing samples at -80 or at least at -20 and minimizing freeze thaw (i.e. through making aliquots).

This is especially important if:

-you are doing any quantitative work

-you are amplifying a rare message,

-your samples are rare / precious

good luck

cDNA is quite stable at both temperatures. However, what you must know is that single-strand DNA sticks to plastic tubes, and it is better to use siliconized tubes when you work with highly diluted first strand, otherwise you may lose it.

DNA is always best stored at 4°C (or -80°C) At -20°C you'll get an "eutectic" mixture where liquid water is trapped in microchannels between solid ice. This concentrates all solutes (including any salts, DNA etc.) and causes shifts in pH and salt concentration that may damage biologicals. We store all our DNA at 4°C, some of it for 15 years (!) without any adverse effect as long as you keep it sterile (of course).

Once time, I stored cDNA at 4°C for three months. However, I found cDNA are good when I used them. Therefore, you can store cDNA at 4°C in one week or longer time.

I would either keep the cDNA at 4°C for the whole week, or prepared the first day, 6 aliquotes of the cDNA, and I would keep all at -20°C. So everyday you thraw only the aliquote that you need. In this way you avoid to froze/thraw everyday, that ruin the cDNA,

IMHO, making the same suggestions over and over again and ignoring all those made by others is not really the point, except for fishing for cheap RG score points.

Thanks Sabine. That's what I told some people in other discussions too. Cheers, Nadine

From my experience on low expressed genes, freeze/thaw cycles degrades cDNA. I usually make aliquots and store at -20ºC. It's tedious if you have lots of samples, but that definitely solved my problems with PCRs. By the way, anyone knows a paper on cDNA stability related to freeze/thawing cycles? Thanks in advance

Please all, let's stay scientific = give a reason for your opinion!

Rules for storage of DNA (clean DNA that is):

1. Never dissolve in water. Pure water has no buffering capacity therefore a solution of DNA will react acidic (desoxyribonucleotide ACID). Acidic soln. will lead to depurination of DNA. Use a slightly basic buffer like 10mM Tris pH 8.0.

2. -20°C is the worst storage temperature of all possible. At -20° what you blieve is solid ice in fact is an eutectic mixture with most of the water molecules trapped in ice crystals and the rest + all solutes forming microscopically small canals filled still with liquid. As a consequence you get a tremendous concentration of all salts/DNA/impurities there and physico-chemical conditions that are uncontrollable. That's why freezing/thawing destroys many biomolecules.

Whenever your DNA is clean (no DNAses, no microbiological contamination) store it at 4°C or if you want at -80°C. I am working for more than 25 years in the lab and my oldest DNA has survived 10 years at 4°C. First degradation was detectable after about 12 years on average and I wouldn't trust it any more after 15 years.

-20° is an emergency measure if you are not sure that your stuff is microbiologically clean. Just as for your steaks at home in your freezer -20 prevents microbiological growth and decay but nothing else. Of course DNA will be a feast for any fungus/bacteria that might have found its way into your tube at 4°C  :-)

So in essence = FIF = fridge is fine

Best

Robert

Doesn't matter how everyone's telling you put into -20, read only Robert's comment. I used to put cDNA in -20 until I realized how inconsistent the cDNA perform over time. at 4 C house keeping genes (HPRT, RPL5 and NDUFA7) amplifies within less than 1% difference for month if not years. One reminder, I noticed cDNA with low expression such as Trypsin-like or Serine protease family and their inhibitors degrades no matter what (relative expression with the same house keeping gene over time). I think we will need a bio-physicist to explain some strong observations for those incidence. 

i have prepared cDNA 6 months back, to avoid freeze thaw cycles i am storing it at 4 degrees. Now my cDNA is absolutely fine. i am still using the same for my qRT-PCR experiments.

Does couple of months of fridge storage apply for cDNA transcribed from miRs? I assume so, but just wanted to check :)

Notably however my cDNA is diluted in water (1:11) as per Qiagen's RT protocol.

I had not realised the issues with -20C storage!

If I am intending to store for a prolonged period would -80C be better than -20C?

Thank you!

Robert K Slany

" As a consequence you get a tremendous concentration of all salts/DNA/impurities there and physico-chemical conditions that are uncontrollable. That's why freezing/thawing destroys many biomolecules. "

I found your post very interesting and I wish to understand it more.

For storing at -20, does the damage accumulate while the materials are stored -20, or does the damage only occur in transition between semi-frozen and thawed?

Would -20 be okay as long as you only freeze/thaw once?