Monday I started the long process of isolating microsatellites de novo for 5 mammal species…. I’ll plan on posting a step by step account of how things are going, so interested people and family can stay up to date. But in addition to this, I hope that this series of posts will serve to fill a small void that exists in the current state of the internet…
For instance, suppose, you do some DNA extraction, and run the resultant genomic DNA on a gel. What is it supposed to look like??? The size distribution of gDNA is a basic measure of the quality of an extraction…. But what is good??? Search the internet currently, and the answer is not easily encountered. This is in contrast to searching for other things (i.e. pornography) where information on the most obscure topics are readily available..
Now more specifically, suppose you are following a well cited protocol, and at each step there is a check, and this check can be run on agarose….but the author doesn’t tell you how it’s supposed to look.. Now that is frustrating! Why does he give you the ability to check your work, but not the ability to interpret the results of that check? You know that there are other people out there using this protocol (a web of science search shows the protocol is heavily cited), but somehow, a google search is simply no help…
Maybe this is exactly how “open science”, “open lab”, “open everything 2.0″ is supposed to work.. I see that Bora has been a champion of this idea, but I don’t see a lot of people heading his words… It seems that these basic types of information should be available… Argh.
Well I will do my part… I will write more as time permits, but for the record, I am using Travis Glenn’s Microsatellite Isolation Protocol (PDF). It is a well cited protocol (Cited ~10 times this year). It uses biotinylated oligos (common msat repeat sequences) and Dynabeads for capture… The 1st step- no surprise is genomic DNA extraction. A couple of points:
- Fresh tissue is preferred, but appropriately stored older tissue is fine.
- You need >50uL of 100ng/uL stock DNA
- Run a couple of uL gDNA on agarose, smearing above 2000bp is OK, but the less smearing the better…
I am using a protocol for DNA extraction that I developed myself…. Here is the PDF. Yield are very good (50uL, >400ng/uL gDNA). It is not quite as clean as using a Qiagen (or similar) kit, but I have never had a reaction not work because of that. It is safe (never have lost a sample), and CHEAP (i.e. basically free)
The gel of typical results….Most of them look pretty good… The sample in the 7th well is not great, and I bet number 8 is a pipetting error….
3 responses so far ↓
1 Laurent // Sep 24, 2007 at 6:36 am
It’s even worse than that. As far as I remember, available information about this specific subject declined over the years… I can’t even find the very basics neither (so that I can give an official reason for students to lurk over the net), while the info was easily found out a few years ago… Seems like web sites happen to die, along with a knowledge nobody cares to maintain a bit further. Internet really has no memory and last short before regular libraries…
2 Anonymous // Sep 28, 2007 at 9:02 pm
Wow, I thought I read that you have a big family. I suspose that is why people dont do this. Eather that or the porn slows them down…ha ha ha. How in the heck do you have the time for all this?
3 Dan McGoldrick // Jan 3, 2008 at 11:22 pm
Well the internet must not be not that bad because I saw your frustration. I have actually met Travis Glenn at the Labratory of Molecular Sytematics at the Smithsonian and have cloned and sequenced microstellites de-novo. Yes - I isolated microsatellites “de-novo” for Pacific oysters - wow! Travis Glenn’s manual was very helpful to me - he’s great. Travis did some really good work with Alligator usats that I learned about, but there are also the red book protocols and Maniaitis’s books - know of them? I think your initial DNA extractions are not too bad based on the gel images (i think that bright band is 600bp? but you didn’t tell the percentage or the marker that you used :-)). One lane with no DNA? For pacific oysters I found the standard proteinase-k / SDS protocol was fine for cloning. Then you will be cutting the DNA with a restriction enzyme and ligating it into a plasmid and then screening? I found that the biotinylated probes for screening have to be well taken care of - do not subject them to dilution or room temps for too long. I used a product from FMC fresh and that always worked. Dupont provided flourescent dNTPS and that worked in PCR reactions for me. Make sure you keep control and care of your cloninjg and screening reagents. All the problems i had getting through making and screening the microsatellites eventually traced back to sloppy reagent histories and concentrations or if screening them in many samples - degraded DNA or primers stored at low concentration in unbuffered solutions. We used to make stock primers and then dilute that 10 fold for a real PCR working mix of primers that lasted about a month. Run controls with the original clone when you test the primers. I also found for every three colonies that I picked, only one ended up providing a useful PCR marker after primer design and testing so don’t get discouraged just pick three times more…
Blah Blah Blah - see! The internet really is a bad thing.
an internet savy concerned and caring scientist,
Daniel J McGoldrick PhD.
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