Pond Boss Forums INTRO Questions & Observations Electrophoresis

This may seem like an odd question but is there anyone on this site who can tell the differece between an electrophoresis DNA analysis of a florida LMB vs that of a nothern LMB?
In my biology class at high school (go cougers!) we just learned how to do DNA extractions and are about to do electrophoresis on some DNA we extracted from srawberries. I asked about it and my teacher told me that if I did some extractions at home I could bring the samples into class for an electrophoresis. Now neither the previous owner who stocked the pond 18 years ago nor the guy who he bought the fish from has ever heard of "florida strain" or "northern strain". They are most likely northerns but since I can't be totaly sure I figured I could catch a bass extract some DNA and get it analyzed.
Only problem is that I wouldn't be able to tell the difference. Is there any obvious indicator between the two that an amateur, like myself, could see or would I have to send a photo of the analysis to an expert?
-Scott

man, i wish my high school biology class had been that interesting. i'm sure you'll let us know how it turns out...will be interesting. mark

Scott,
I can probably get it looked at by GA DNR if you can get a copy. I'm sure a few of them can tell since they did a study on bass genetics in GA waters. I did not get to perform an electrophoresis until my third biology class in college, impressive.

Scott I did a similiar thing in high school biology...how was yours done? I can't remember what DNA we looked at but it was very interesting. However, I do remember that each group in our class had a different result. Im not sure if that was due to the inprecision of our instruments or what. Good luck.

Chris

First what we did was take the strawberries and smash them up in a zip lock bag to break up the cell walls. Then to help dissolve the cell membranes we mixed in a buffer that was primarily diluted shampoo (the cheapest source of Acetal Laurel Sulfate), somtimes meat tenderizer is also used in the buffer to help destroy enzymes that might damage the DNA. Next we filtered the mashed strawberry cells through a cheese cloth to remove all the fibers and cell wall chunks and organells and any other solids leaving mostly fluids and DNA. These fluids were put into a test tube and very pure, freezing cold alcohol was gently poured in above it. Now since one end of the DNA molecule is attraced to water (hydrophillic) and the other end is repelled by it (hydrophobic) the DNA gathers and forms fibers right where the fluids and the alcohol are stratified. If you stir it gently or blow bubbles into the bottom using a small straw or pipette, the hydrophillic ends of any DNA the moves up into the alcohol will sort of turn in on each other also forming fibers with only the hydrophobic ends exposed to the alcohol. It's really easy to see the DNA fibers suspended so all you have to do is take a freezing cold glass rod or something and gather up the DNA like cotton candy. Strawberries were used because they have been altered to have 8 times the natural amount of cromosomes but the process is just the same on anything else.

To do the electrophoresis first you take special restricton enzymes that cut the DNA at specific points along the DNA code. for example EcoriII cuts DNA wherever there is a GAATTC. If the DNA code is different it will make different amounts of different sized peices and if it is the same it will be the same. Then you prepare these thin gells with little wells at one end. you put the cut up DNA in the wells and then attach the electrodes with the negitive end just behind the wells and the positeve and the other side of the gell. The current carries the DNA pieces through the gell, the little pieces move farther and the big pieces get caught up in the gell and don't move as far. Where it stops you get a dark band. The same DNA will always produce the same series of bands depending on the enzyme used.
Some genetic traits produce what they call "markers" on an electrophoresis, visible bands that wouldn't normaly be there. I was hoping there was some obvious marker for either florida or nothern.
Oh another note, I was wrong we wont be doing the electrophoresis untill march. So it'll have to wait a while untill I can have any results.

-Scott

sounds like you have a budding career ahead of you. good luck. it's funny you mentioned the strawberrys in your analysis....my genetics professor in college was the guy who genetically engineered the apple sized strawberry...but maintained the flavor of the smaller strawberry... who knows...maybe you'll be genetically engineering a 40 pound largemouth someday!!

Scott,
The veterinary college at Texas A&M University has plenty of comparables for you. They have a lab specifically to assist the consuming public. Here in Texas, when we want to look at genetics for largemouth bass, that's where we go. I bet, if you give them a call, they will help by looking at your results, or by providing copies of their work.
Great stuff.

Hey thanks guys. Really great info.
I do have a few follow-up questions
Would it cost anything to get help from Texas A&M?
Do they have a website or something where I might easily acess some of thier research?
Do you know if there is anything else usefull to be learned from a DNA analysis, like how badly my bass genepool is inbred and in need of some new genetics?
If so, could I tell from a single sample or would I need to sample a whole lot of bass?

Also, I'm home sick today with nothing to do so this morning I decided to try an extraction, but it didn't go as well as I had hoped. Everything went just fine until I added the alcohol. I could see the DNA precipitating and there was actualy more than I had expected but when I went to gather it up it didn't collect together in long, sticky fibers. It just floated around in little bitty chunks. I finally gave up trying to pick it up and just skimmed it off the alcohol with the suspended bits of DNA in it. I figured since you store it in alcohol anyways it couldn't be all that bad that I couldn't seperate them but I cannot figure out why the DNA wouldn't gather together.
The best explaination that I can think of is either that it took too long to chop up and smash the fish meat into goo and having not used any meat tenderizer that enzymes dammaged the DNA in some way. Or more likely that the 91% Isopropyl Alcohol 9% water solution that I used was simply not pure enough.
Have any of you experienced something like this before or have any idea what might have happened?

thanks
-Scott

Oh, I almost forgot.
I have a chemistry question too.
(this may be an odd question for a pond management website but we've got guys from all walks of life here so what the heck)
I've been trying (with no success) to purify my alcohol-water solution by getting it cold enough that the water would freeze, the alcohol wouldn't, and I could just skim off the ice leaving a more pure alcohol.
Is something like that possible?
If so how cold would it need to be?
If not is there another way?
If there is no easy way to purify it and I need to buy a more pure alcohol, deos anyone know exacly how pure it needs to be?

-Scott

hey scott i might be able to help you out some, but i'll need some more info...

What kind of alcohol is it that your working with?

Also what type of water are you using? Tap? Distilled?

I'll check back later and try to answer as best as I can.

Chris

jr. brockey, if i can recall from organic chemistry, we used ice and salt (rock salt) to acheive colllldddd temps. (same concept as when you make home ice cream with the ice and rock salt on the outside of the container). was this what you were asking about? also, how about dry ice/liquid nitrogen?

also, pharmacists use pga/everclear for compounding (mixing) drugs/pharmaceuticals. hopefully, you have little experience with pga/everclear!!! you will have to ask your dad to purchase it and monitor it. i remember when i finished high school, i worked in a pharmacy... i noticed a bottle of everclear alcohol under the pharmacists work station. i didn't have much experience/knowledge of alcohol other than folks drank it and could get drunk!! well, one day i noticed the bottle, then the next day there was a little bit gone, the next week some more gone, and the end of the month, the bottle was almost gone!!! well, the pharmacist was a friend i've known since i was young...so i innocently asked him with concern..."do you have a drinking problem at work, because i notice the everclear bottle underneath your work station seems to be almost gone...can i be of any help, do you need help??"" !!!! he laughed, and said i was nice kid for being concerned...but he then informed me and showed me how it was use to compound medications!!! anyway, funny story i think.

keep us posted, mark

could you try and distill the alcohol to make it more pure?

tritonvt, it was regular rubbing alcohol. The bottle said it was 91% Isopropyl Alcohol and that the only other thing it contained was water.
Mark, great story with the everclear dude. Even being in the freezer and doing the ice and rock salt trick at the same time didn't get it cold enough. And I think that going to the extent of using liquid nitrogen or trying to distill it might end up being more costly and difficult than just trying to buy a more pure alcohol (but I have always wanted an exucuse to play with some liquid nitrogen ). Besides I don't know what the minimum percent alcohol for a DNA extraction is, so I can't be sure that purity was the problem. That was just my best guess.

-Scott
side note for mark: I think you kinda got the name mixed up. its not J Brockey, its more like J.B. Rockey. You see, my dad's first and middle initals are J.B. (wich is what he usually goes by) and the Rockey is derrived from my mom's name, Racquel. Not extremely important, but I figured you might like to know.

well scott i tried to do some research but came up with nothing. i did see a site that said the freezing point of rubbing alcohol is around -88 C. what was your technique in trying to freeze it. im not really all that familiar on how alcohol reacts with water, but from other things i've read it acts the same as 'salt' does whenever applied to roads in the north. it either lowers the freezing point. therefore im guessing that the alcohol is acting as though it is dissolved and therefore you might not be able to freeze of the water. i dont know for sure though. i know that alcohol has a lower boiling point than water so that would always be an option to consider....but remember alcohol is very flammable. try some searches on distilling alcohol or purification on alcohol and see if you can get anything.

Chris

I do believe what you are looking for is the pure Isopropyl alcohol. I did a search and found two other versions, 70% and 99.9%. I would think a pharmacy could sell you the purest version.
Wood

jb ... it may just be a good idea to call your local organic chemistry professor at a nearby college or your high school teacher may know. even though you are not in college...an organic chem professor that hears about a high school student interested in learning...most of them would be glad to help... just say "cool" "neat" and "wow" and "you are so smart" alot...they'll tell ya whatever you want!!! mark

I work in the biology department at my university. We have a chemical stock room, and in the flamables room is a 55 gallon drum of 100% ETOH, which I believe is the same as or similar to isopropyl. If I were you, I would buy pure isopropyl. I know you can get it in 16oz bottles too. We have lots of those, try contacting your local U, they may not be able to give you any but would be able to put you in contact with a wholesaler who should.

I just now realized that I never updated this post.
I found out what the problem was. The isopropyl apparently deos not repel the hydrophillic ends of the DNA strands so strongly as Ethanol deos. My biology teacher said that if it were 100% isopropyl it MIGHT work but ethanol only as strong as 91% almost certainly would. So everclear or somting like that is definatly the way to go next time.
-Scott
(sorry for the delay, I was just sure that I had already let you guys know what was up)