After picking colonies, preparing DNA samples from them, digesting a small aliquot of the plasmid prep with the appropriate restriction enzymes and preparing an agarose gel ... you end up loading your minipreps instead of your restriction digests.
Damn.
- Log in to post comments
More like this
The general steps in genome sequencing were presented in the earlier installments ( there are links at the bottom of the page), but it's worth repeating them again since each of the earlier steps has a bearing on the outcome of those that come later.
These are:
Break the genome into lots of small…
Astute ERV readers have noticed a couple of odd things about my research.
1- How the hell am I cutting and pasting bits of a retroviral (RNA) genome together?
2- How the hell do I have a (seemingly) endless supply of HIV-1 for my experiments?
The answer to both questions is, infectious molecular…
OK I finally did that experiment that people asked for ... and more.
If you want to know why I performed this experiment, read this post on RNA treatments for autism. Here I present to you evidence that your body is secreting enzymes, called RNAses, that will chew up your RNA in minutes.
So the…
7AM, up out of bed. I prepare breakfast, two slices of focaccia with salmon and olive spread with an espresso to wash it all down. I quickly flip through the paper. For once the NY Times Science section has an article on some basic molecular biology (it must have been the evolution angle). After…
*%&*$&£%&$!!
You kept the plates, yes? At least then you've only lost a day ;oP
my sympathies
you could always gel extract...
been there done that.
It could have been worse. You could have painstakingly purified some protein over two days, and then, in a last step, added 10% SDS to the protein, instead of the 100 mM NaCl from the bottle sitting next to the SDS bottle (yup, I actually did that once), all because you were trying to do 10 experiments in the day.
What about purify an antibody from 20ml of serum using sepharose A beads, elute, bind to the antigen column, elute, concentrate, and then, when spinning down the 40ul you get in the end, the tube brakes in the centrifuge!
*** UPDATE ON THE TERRIBLE EXPERIMENT ***
What I wrote in this post happened two days ago. Here's what's occured since then.
Poured another gel, ran the restriction digests anyway (I had saved some bacteria), all the colonies had insert but 7/8 were ligated in the opposite orientation. I grew up leftover cells from that one "good" colony, prepared another DNA miniprep, sent it for sequencing. I just got the sequence back and ... it turns out that this last colony had the insert backwards as well, but it had a point mutant in the restriction enzyme site that I used to determine the orientation.
AARRGGHH!
Just tell the PI you got a little carried away with undigested controls. And now the backwardly-inserted fragment is a backward-insertion control. See? Failed experiments are really just insightful controls in disguise.
8/8 in the opposite orientation? Sounds like might have yourself a pretty toxic insert there. Let us know if you can get the right clone in the end. Else you may have to use a less leaky promoter or lower temperature or something..
Oliver,
I just picked 20 other colonies.
Yeah it's either the translational product or the RNA itself - I'm testing whether a 5'UTR is doing something funny - it looks like this stretch of RNA has some massive fold. My huntch is that the RNA is toxic depending on what is directly upstream of this UTR.
Another fun thing to do is to try and sequence a genomic dna sample rather than the PCR product of interest. That;s my fun experience from last week.
Yup, toxic inserts are frustrating. I haven't come across problems with RNA yet, but in our lab we often have problems with eukaryotic genes forming toxic inclusion bodies under constitutive promoters and I once had trouble cloning an enzyme that acted on a E. coli central metabolite, shunting it into a metabolic dead end. The solution was using a non-leaky promoter.