SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Sample Prep / Library Generation (http://seqanswers.com/forums/forumdisplay.php?f=25)
-   -   Covaris microTubes or 96 well plates - alternatives? (http://seqanswers.com/forums/showthread.php?t=19118)

gajz 04-11-2012 06:31 AM

Covaris microTubes or 96 well plates - alternatives?
 
Hello,

I am using Covaris sonication method to fragment genomic DNA. The only issue I have so far is that the microTubes are quite expensive ($5 each), as well as the 96 well plates ($ 450 each).

Does anyone have tried different glass tubes or plates that are more cost effective? I am not planning on reuse them, just want a cheap alternative.

Thanks a lot,

Heisman 04-11-2012 06:41 AM

We use PCR plates regularly. The sonication is not quite as good (ie, harder to get 150-200bp and a tight distribution, and we sonicate overnight, but it's obviously much cheaper. I would try a plate (we've tried one type and it works for us... I bet others would work too) with commercial genomic DNA and see what happens.

gajz 04-12-2012 05:44 PM

Thanks for your answer Heisman,

I will try with a 96 plate to see what happens, but do you think that the fact the wells are made of plastic can affect the results? How important is doing the sonication on a glass surface?

Heisman 04-12-2012 05:46 PM

As I said, it's harder to get down to the proper base size, likely due to that reason. I would try some sessions sonicating for 10 minutes with 10/5/500, 10/5/200, 20/5/500, and 20/5/200 settings and see how that performs, then go from there.

Hamid 04-18-2012 10:07 AM

1 Attachment(s)
Dear Gajz,

I apologize for the delay in responding to your post.
While seemingly beneficial to use a low cost PCR tube in your Covaris instrument, it is critical to address the energy requirement to generate small DNA fragments. Covaris specifically engineered the microTUBE to work in conjunction with AFA to control the energy for generation of small DNA fragments:

1. The amount of energy required to shear DNA is inversely proportional to the size of the DNA fragments desired. For example, large fragments such as 3kb require low energy and consequently, plastic may be used (e.g., Covaris miniTUBE). More importantly, however, is that to generate small fragments of DNA, a high level of energy is required. If this energy is not efficiently transferred to the sample and if the subsequent heat is not readily dissipated the sample will quickly heat. As a result, for shearing to NGS-size fragments it is essential to maintain an isothermal processing temperature during shearing by controlling the thermal events through BOTH hardware (Covaris AFA) and the consumable (microTUBE). These tubes are specifically designed to work with the acoustic circuitry to maintain an isothermal environment during DNA shearing

2. Plastic materials such as polypropylene PCR tubes absorb acoustic energy and then through convective heat-transfer your sample is heated. Your sample may be in an iced water bath, but your internal sample fluid is heated. The heat is then prevented from efficiently dissipating from the sample since the polypropylene then acts as an insulator. I have attached a slide of the temperature mapping of DNA samples sheared to 200pb in the Covaris microTUBEs compared to the same sample processed in a think wall 200ul PCR tube. As you can see, even though the PCR tube sample is in a 6C water bath, the temperature of the sample is greater than 50C. In comparison the temperature of the sample in the microTUBE using the same settings is the same at that of the surrounding water bath.

3. The lack of the thermal control when using the PCR tubes/plates will certainly effect reproducibility, shearing size distribution, total DNA recovery, and will cause thermal biased shearing of samples in regions with low GC content.

4. Several large sequencing centers did initially evaluate using PCR plates instead of the microTUBE plate, but observed shearing inconsistency, and failed sequencing runs which they attributed to the shearing in the PCR plates.

The microTUBE is an integral component of the AFA circuit.

Thank you

Hamid

tinkma 02-12-2018 07:30 AM

Hi Gajz,

Did you ever test shear using PCR plates? How did it turn out and may I ask what offest did you use? Thank you.

seq198 04-25-2018 05:15 AM

You could consider enzymatic fragmentation (cheaper and faster than Covaris). Nearly all NGS kit provider have an option for enzymatic fragmentation now. The FS kit from NEB is promising in our hands, though we have sequencing data so far.

NGSbirb 06-12-2019 08:29 AM

Hey all,

I'm late to the game, but what about using glass-bottom plates? These would be similar to the microTUBE plates (borosilicate glass) and come at a dramatically cheaper cost. Something similar to 96-Well Krystal Glass Bottom Imaging Plates from Porvair?

luc 06-12-2019 04:51 PM

Covaris does now sell plastic plates and strips for shearing on their very latest sonicators:
https://covaris.com/products/afa-tub...ials/afa-tube/
They are a tiny bit cheaper.


Quote:

I'm late to the game, but what about using glass-bottom plates? These would be similar to the microTUBE plates (borosilicate glass) and come at a dramatically cheaper cost. Something similar to 96-Well Krystal Glass Bottom Imaging Plates from Porvair?
You definitely don't want them to break during sonication.

NGSbirb 06-13-2019 11:43 AM

$398 for 1 plate still is so expensive. Definitely don't want them to break during sonication. Unless there's some magic to the covaris glass, a different plate should work (sans the AFA fibers)....

Thanks for your reply.



Quote:

Originally Posted by luc (Post 226807)
Covaris does now sell plastic plates and strips for shearing on their very latest sonicators:
https://covaris.com/products/afa-tub...ials/afa-tube/
They are a tiny bit cheaper.



You definitely don't want them to break during sonication.



All times are GMT -8. The time now is 09:56 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.