EZ Bead experiences

[epistatic]

I found out we are getting a SOLiD 5500. I have heard mixed reviews of the new EZ bead system, the sales guy even said the early customers had problems. I am not sure if he is setting low expectations for a product that does not work well or if it has improved. Any input from experienced users is greatly appreciated.

[chenglei]

Our lab has 3 sets of EZbead at Hongkong. Most problems mainly come from the Emulsifier, the oil master mix made by this machine is not stable for reaction and make the WFA result very poor.

[VanessaS]

We have two ezbead systems, love them and swear they are the best money ever spent. I've had zero problems with the machines.
Make sure however, that until further notice, you are using the modified protocol for making the oil, where you use syringes and measure by volume not weight. My understanding is that the weighing protocol is highly dependent on your scale and many people had problems with the new protocol, so AB now has a protocol where you take the kit and make 6 tubes of the oil like we used to do before EZBead. I do that, vortex then combine and throw on the emuslifier for good measure.
Since doing this we've never had a problem and get fabulous, consistent beads from the EZBead system.

[epistatic]

That is great to hear, thanks!

[pmiguel]

I heard of an instance where 50 ml conical centrifuge tubes used to mix or hold some reagents for EZ Bead emulsion were cited as the culprit for poor results. (Possibly problematic "releasing agents" coating the interior of the tubes detrimental to the emulsion?)

So, that said, maybe someone getting good results with the EZ Bead (VanessaS?) could tell us what type of syringes they are using to measure oil in the modified protocol?

--
Phillip

[VanessaS]

We use the Falcon 50 ml conical tubes and the BD slip tip syringes.

[VanessaS]

For those of you not getting good results, is your emulsion broken after ePCR?

[pmiguel]

Hi Vanessa,

We have only done 2 runs on our EZ Beads -- both the same library. We have not had any EZ Bead training, so I'm not sure we would recognize broken emulsions. In plates you look for "freckling" at the bottom of the wells. Not sure what the equivalent would be for a giant bag of beads.

--
Phillip

[VanessaS]

Its kind of the same but on a bigger scale. You would see a line of the beads at the bottom and a bit of a gradient going up from there. When its not broken, and is just freshly done, its a nice pale tan-peachy color and is uniform.
If you let it sit over night it does separate a bit but doesn't have the bead dropout.

[pmiguel]

For those of you not getting good results, is your emulsion broken after ePCR?

Hi VanessaS,
I spoke with someone yesterday who saw an instance where an EZ Bead was not giving good results. The emulsion was broken. It was tracked down to 50 ml conicals being used, as I mentioned above.

--
Phillip

[VanessaS]

Do you know what brand, so everyone can steer clear?

[pmiguel]

I don't know, but probably best to only purchase the ones recommended by AB -- I believe those are the Falcon tubes that you use.

[PersianExcurzion]

But how long?

I see that AB claims about an hour of hands on time, but how long does the total run take, and is the hands on time all at once, or is it spread out between steps of each machine?

Thanks!

[epistatic]

I would calculate over an hour of hands on time for the emulsifier and enricher. The amplifier is just hanging a bag in a sideways t-cycler.

The quick reference guides will give you a good idea of actual effort required for each step. The EZ bead system is the use of the Covaris, Emulsifier, Amplifier, and Enricher. Lots of fun compared to the cBot.

EZ Bead Emulsifier
http://www3.appliedbiosystems.com/cm...cms_083200.pdf

EZ Bead Amplifier
http://www3.appliedbiosystems.com/cm...cms_083201.pdf

EZ Bead Enricher
http://www3.appliedbiosystems.com/cm...cms_083202.pdf

[VanessaS]

Hmmm. So you gotta make the oil, that takes 15 min or so, and have to let that degas for 30 min. You have to defrost your ePCR stuff, which can be going at the same time. You need to dilute your library, so however long that takes you. Prepping the bag takes about 20 min after the oil is ready. Making the emulsion is 15 min.
I'd say its a fair statement that it takes an hour of prep time to get things onto the amplifier. Thats like a 3.5-4 hour run, which can be done overnight. It takes about 10-15 min to set up the enricher including transferring the beads. The enricher takes another 4 ish hours.
Its all easy-peasy and makes life in the lab just a bit more rosy.