I am doing practice runs of an ES cell lysis protocol that I am planning on using for southern blot analysis.
I get the standard spidery precipitated DNA after addition of isopropanol, I spin it down and resuspend in ~30 ul tris buffer. It goes from a compact nice white pellet to this cloudy goop which is literally a single gooey entity. It's only pipettable using a wide-bore 1 ml pipette, all others get stuck and don't work.
I highly doubt it is protein contamination, as the pellet I get is a super nice, small dense one. My question is, I would love to do PCR screening first before southern blots, but how on earth are you supposed to pipette a micro-liter of goop that doesn't even detach itself?
Lysis buffer: 100 mM NaCl, 50 mM tris, 10 mM EDTA, .5% SDS, 5 mg/ml PK. Digest O/N at 56C.
Precipitate with isop->spin down, wash x3 with 70% EtOH, resuspend.
Any help?
Your DNA is probably too concentrated, just add more volume of your resuspension solution and may be incubate your samples at 37°C for some time.
That pretty normal for genomic DNA. These are really big molecules and as long as they are intact, they form this higly viscious substance. You can either try to dilute your DNA or shear it to some degree to get better solubility. The later version bear the risk, that you cannot amplify your target sequence.
Thank you both for your answers.
Indeed, I figured it was concentration, however, serially adding more and more buffer until some part of it wasn't viscous anymore yielded me DNA at around 50 ng/ul.
Incubation at 37 o/n seems to not have helped unfortunately.
Since these are in 96 well plates, I had originally wanted to just take 1ul from each well, do a quick pcr screen and then dump restriction enzyme mix straight into each well for a southern.
I agree with Christian that it's probably just how big the macromolecules are, but how do you use this for PCR?
I imagine after a digest it would be nice and no longer viscous, but then I can't pcr off of it :p
You can break DNA simply by pipetting up and down or by using a sonicator. Be careful though, because when your fragments are getting to small, you will not be able to amplify them by PCR.
Sounds like you are getting lot of DNA from these preps. If you want to use these for southerns I would stay away from anything that might shear it (ie: vortexing, sonication, excessive pipetting), or you are going to get smeary bands for sure. Make sure that you aren't letting the plate dry out too much after the 70% washes, this can make pellets hard to resuspend. You might want to try just spinning the plate to pellet all the goop at the bottom of the wells, and then transferring the sup to a new plate for your PCR and eventual southern digest - you will definitely have a high enough concentration in the sup for PCR and may have enough for your southern as well.
When I do large plasmid preps I usually get some insoluble goop that is not responsive to any amount of heating, vortexing, or trituration. I just pellet it out of the final sample. Not sure if it's all DNA or carryover of contaminants from earlier steps but my yields are fine without it.
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