I spend much more time teaching and writing, than doing actual benchwork, these days, but occasionally I make forays into the lab for collaborative projects. Today’s task was to start the genotyping process. I’m expanding two of my lines of knockout mice, to complete a few remaining experiments for a manuscript in preparation, and of course I need to know who is carrying the targeted null allele, and who isn’t. Actually, I need to check for the presence of two targeted null alleles, which in this strain should be present in cis, on chromosome 11 (shared synteny with chromosome 17 in humans). Presence of one targeted allele and not the other = very annoying, as this usually means that re-recombination has occurred.
As you’ll see, my method is perhaps a bit primitive by current standards (the protocol dates from the early 1990s), but I get clean results every time, a great DNA yield, and the process is relatively inexpensive. First, one needs to obtain tissue samples (ear punch or tail clip) from which the genomic DNA will be isolated. Because I’m more than a little obsessive compulsive, the cage cards and Eppendorf tubes are color-coded for each knockout strain. Mating cage, litter number, and ear punch number are also written on each tube, and of course there’s a master list in the breeding book.
OCD much?
For the next steps, I unlax‡ the OCD a little, and put 4.0 ml of lysis buffer into each of those tubes lying off to the left. Now, those of you with delicate or compulsive constitutions may want to skip over this next bit ….
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I never, ever weigh out the proteinase K, but instead dip the large end of a yellow tip into the powder, and stick that into the tube with the lysis buffer and swirl until the proteinase powder falls off or dissolves. Ugly, I know, but it works every time. Probably because I have turned it into a ritual.
Vortex the solution briefly, and add 200-500 microliters to each tube (depending on the size of the samples). Make sure the lids are snapped tight, and that there are no flaws in the Eppendorf tubes, because the tubes are going to shake and bake horizontally, in this contraption:
Never underestimate the usefulness of colored time tape. EU non-compliant date optional.
Off we go to the environmental shaker overnight ….
‡ My thesis mentor claimed that I could not relax, because I had never been lax at any time. Therefore, unlax. The (il)logic of this will likely give you a headache, so I suggest avoiding attempts to puzzle it out.
A Mysterious Mystery
I received a very nice surprise today at work:
A mysterious mystery, today
I only know that:
- The package was sent from Gloucestershire.
- The colour pencil box has a sticker from Coln Gallery in Cirencester on it.
- Dr. Etchevers has been mysterious recently.
- I can’t read the signature on the Customs Declaration, and neither could my lab neighbors.
All I can say is “THANK YOU!ELEVENTY” to whomever(s) sent me this lovely and generous gift … rest assured that I will put the coloured pencils to good use, defacing altering funky old natural history books and scribbling pictures of birds and flowers and seed pods.
Last updated:
Tuesday, 16 Jun
2009 - 00:15 UTC
A lovely colourful post, even without that great surprise at the end! How I love packets of crayons and pens….
;-) Hey, thanks for the genotyping tips and for the nice colourful Eppis.
And about the surprise, it seems to me that your old books will be much vivid now… :-) (btw. I love pens and pencils in general and got new Faber-Castell a few months ago… black & white though).
haha, I was about to write “your not OCD since I do the same thing” … then I thought about it ;)
I did the same mix of writing on tubes and keeping master list and ear tag number etc… although we normally don’t trap the eppis in between two racks but I leave them in 55C shaker over night, standing in a nice little rack by themselves. (that’s tail extracts though).
Hope your PCRs go well too!!
That does sound lovely, to be a patron of the arts from the Cotswalds.
I’ve no problem with a pinch-of-that style cookery in the laboratory, but my personal compulsiveness lies in folding over the edge of the colored tape such that one has a neat border with which to remove said tape sometime in the future. Be it on pipetters or on glassware.
And if your dog dispatched of small furry things without making them suffer for enough time for you to try to intervene, well, what else can you do?
@ Maxine and María José: I love pens, pencils, crayons, and art supplies in general. Walking into an art supply shop, like Texas Art Supply in Houston, is visual and olfactory paradise.
@ Åsa: These were, in fact, tail extracts … sometimes a Southern blot is required to confirm PCR genotyping, and it’s nice to have enough DNA to go back to, just in case. My collaborator will do the PCRs this time. Hooray!
@ Heather: You are still being mysterious! :-D I usually fold the tape under as well, because I hate having that gummy stuff stuck under my fingernails, or on gloves.
Dogs will be dogs, I guess, and as a friend pointed out, I’m not sure she had a choice in the matter. It is, after all, her backyard, and I can’t punish her for behaving instinctively. It’s just a matter of time before the dog gets skunked, though … better have the de-skunk ingredients on hand.
UPDATE: Test-drove the lovely Caran D’Ache this weekend in the altered book; plan to have photos of the results posted later today ….