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Old 06-14-2012, 10:54 AM   #1
Smriti
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Default Purchasing Human DNA (genomic)

Hi There,

Can anyone recommend me some vendor to buy Human DNA (intact genomic)? Once I bought Human DNA from Coriell Institute and that did not turn out to be a good quality DNA.

Quick response would be appreciated as I am stuck in middle of my experiments.

Thanks!
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Old 06-14-2012, 11:45 AM   #2
Heisman
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We've had good luck with Promega.
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Old 06-15-2012, 04:21 PM   #3
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Thanks Heisman for quick response!

Also while preparing dilution of genomic DNAs (Ecoli or human), how do you get homogeneous sample? Do you heat it around 37-45C everytime before using DNA for library prep? I observed lot of variations in concentration of stock solutions using Qubit BR assay. I assume its bec of inhomogenity of stock. And also, is homogenity maintained once you heat it and store it back in freezer?
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Old 06-15-2012, 06:21 PM   #4
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You can heat it or leave at the bench for a couple hours but I would definitely avoid heating and freezing it. That will just fragment the DNA. Aliquot the DNA and don't freeze an aliquot more than a couple times.

If you or someone near you has access to a cell culture facility you might be better off just extracting DNA fresh. Its cheap and easy.
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Old 06-16-2012, 10:59 PM   #5
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Thanks genericforms!

I mostly aliquot stock genomic DNA after heating, diluting and freeze it. One by one I use the aliquots by transferring them in fridge for limited time. However, I observed these stock aliquots show variation in concentrations after few weeks, so was wondering if they need to be warmed at 37-40C before use?

Definitely, extracting DNA fresh is the best solution but right now we have only option of buying DNA from vendors.
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Old 06-17-2012, 09:35 AM   #6
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Yes you should warm them. If I leave genomic in the fridge for a long time I see the same thing. Sometimes you can even get precipitates. If you do you can always do an ethanol precipitation and reconstitute it again.
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Old 06-17-2012, 11:35 PM   #7
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@Heisman, which genomic (human) DNA have you used- male/female or generic? Have you done genomic library construction using it? Also, on website it says storage at 4C, did u store at 4C or -20C after receiving it?
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Old 06-17-2012, 11:37 PM   #8
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I think we get generic and we keep it at 4C as it says. I've used it for library preps (to practice various things) and it's yielded good results.
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Old 06-17-2012, 11:46 PM   #9
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Ok Thanks for this info! another quick question that I have been asking others, how are you maintaining homogenity of your prepared stock? Dont you see variation in concentrations when you store it for long at 4C? In what concentration range, you dilute your stock and store? I am facing big time problem with storage of my stock Ecoli.
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Old 06-17-2012, 11:48 PM   #10
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I vortex vigorously before using and otherwise do not heat it up. I get fairly consistent concentrations. However, it's almost always a bit less than what's specified on the tube. I haven't received it fresh (others in the lab have) so I'm not sure if it comes more dilute than stated or if somebody in the lab messes it up somehow or whatever. But it's consistent after that.
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Old 06-17-2012, 11:53 PM   #11
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Thanks! Dont you see slight shearing of this genomic DNA by vigorously vortexing? And do you test your stock from time to time, to see if its sheared while kept in fridge, or for your applications, you do not worry about DNA getting slightly sheared? Which instrument do you use for shearing DNA in your sample prep steps?
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Old 06-18-2012, 06:11 AM   #12
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I've never tested it that rigorously; it's always performed well enough for our purposes (testing library preps/PCR conditions, etc).
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Old 06-18-2012, 01:34 PM   #13
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Quote:
Originally Posted by Heisman View Post
I vortex vigorously before using and otherwise do not heat it up. I get fairly consistent concentrations. However, it's almost always a bit less than what's specified on the tube. I haven't received it fresh (others in the lab have) so I'm not sure if it comes more dilute than stated or if somebody in the lab messes it up somehow or whatever. But it's consistent after that.
I am not a fan of vigorously vortexing the sample as it can cause degradation. Same with freeze/thaw. However I imagine that vortexting will yield a homogenous mixture.

Pipetting up and down should work okay as long as they are not precipitates.
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Old 06-19-2012, 07:17 PM   #14
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4 degrees is the point where water starts to expand as it gets colder rather than shrink which means the ice crystal structure is starting to form and break (remember temperature is just an average). This will slowly cause the DNA to be excluded from those regions and group up. So even storing in the fridge will not maintain homogeneity for long.
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Old 06-20-2012, 11:59 PM   #15
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So, what are the conclusions from this thread: we should store DNA aliquots in -20C and whatever present batch we are using in fridge. Also, to homogenize the DNA sample, we should either do gentle pipetting or gentle vortexing. Can tapping the tube with fingers also homogenize DNA?
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