Spoggly Isay P's Profile

If you're not part of the solution, you're part of the precipitate.

May the torque be about you.

it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep

Ramazzotti G., and W. Maucci. 1983. The phylum Tardigrada--3rd edition. English translation by CW Beasley. Mem. Ist. Ital. Idrobiol. Dott. Marco de Marchi 41: 1B680.

5 enzymatic defects have been demonstrated or deduced in Zellweger syndrome

Eosin Methylene Blue (EMB) Agar. EMB agar is used
for differentiation and isolation of gram-negative bacilli from mixed
populations of bacteria. EMB agar employs a combination of the dyes
eosin and methylene blue as an indicator which gives a distinct
difference between colonies of lactose fermenting organisms and those
which do not ferment lactose. Lactose fermenting colonies are either
dark or possess dark centers with transparent colorless peripheries,
while organisms which do not ferment lactose remain uncolored. This
purple color is due to the absorption of the esoin-methylene blue
complex which forms in the presence of acid. (However, EMB should not
be used as the sole indicator of lactose fermentation. It should be
used in conjunction with MacConkey agar or lactose fermentation
broth). Certain members of the coliform group, especially
Escherichia coli, exhibit a greenish metallic sheen by
reflected light. Enterobacter-Klebsiella colonies are larger,
usually mucoid, occasionally confluent, and with dark blue centers.
Gram-positive organisms are inhibited by the dye content of the
medium making this medium selective for gram-negative bacteria.

Simmon's Citrate Agar. This is used for the
differentiation of gram-negative enteric bacteria on the basis of
citrate utilization and is part of the IMViC test. Only those
organisms capable of utilizing citrate as a sole source of carbon
will grow on Simmon's citrate agar. These organisms are able to
cleave citrate to oxaloacetate and acetate via the citritase enzyme.
Another enzyme, oxaloacetate decarboxylase, then converts
oxaloacetate to pyruvate and carbon dioxide. Carbon dioxide combines
with water to form sodium carbonate, an alkaline compound. As a
result, the pH of the medium rises and the indicator (bromthymol
blue) changes from green to blue. Coliforms either do not grow at all
on this medium or grow so sparsely that no change is reaction is
apparent. Organisms are streaked onto a slant of Simmon's citrate
agar and the reaction read after 24h of incubation.

VP test determines if neutral products such as ethyl alcohol, acetoin
and butanediol are formed.

Base-catalyzed hydrolysis of DNA gave four nucleoside products, which proved to be N-glycosides of 2'-deoxyribose combined with the heterocyclic amines.

Ethanol Wash
Ethanol washes are performed after salt/EtOH precipitations to remove any residual salt from the nucleic acid pellet. The wash employs 70-80% EtOH which will solubilize salts but not nucleic acids.

Do
Add 70 - 80% EtOH to the nucleic acid pellet. The volume should be sufficient to at least cover the pellet and wet the sides of the tube when vortexed (there is no volume too large). Vortex the sample for 1 minute; the pellet should come loose from the tube and be broken up in the EtOH. Centrifuge the sample 10 - 30 minutes, to recollect the pellet. Aspirate off the EtOH.

Don't
Don't just add the EtOH and immediately decant. The pellet should be vortexed so that the EtOH can penetrate the sample and solubilize salt.

Don't forget to respin! The pellet must be firmly reaffixed to the tube so that it is not lost during aspiration.

What does the alcohol do? Why does the DNA rise
to the top after adding alcohol? (DNA will not
dissolve in this alcohol, so the DNA comes out of
the solution, or precipitates. It is less dense
than water or cell scum--which is what settles to
the bottom of the glass--so it floats up into the
alcohol layer, where you see it as a snotty, string-like
substance, with small bubbles formed on it.)