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Trifluoroacetic
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DNA Extraction and Electrophoresis
Hi guys I've been on here before under a different name but it's been a while and I forgot my user ID and password.
Anyway I thought I would let you know that I have been doing some DNA extractions. I am in the process of experimenting with running the DNA through
an electrophoresis unit and staining the DNA bands with various Dyes.
I made my own gel tracking dyes and buffers and am using Agar ager from a food mart.
I do have a company that has sent me a bunch of free supplies and I do have the ability to order real enzymes, ethidium bromide, buffers, and agarose;
but at this time I am using the supplies I have on hand.
right now I am experimenting with staining DNA with Nile Blue and Methylene blue Chloride. I have a bunch of pictures if anyone wants me to post them.
Just let me know.
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Trifluoroacetic
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DNAExtraction
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Trifluoroacetic
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DNAExtraction
Here is a test run of my homemade tracking Dye.
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mac
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Hi Trifluoracetic,
This is my first time at the sciencemadness forums - they seem great! I just wanted to let you know that some friends and I are trying to develop and
publicize (via instructables and videos at diybio.org) a DIY gelbox that is pro-grade and includes a transilluminator (blue LEDs).
We posted a fun DNA extraction instructable a week ago, and when we get the gel box working we'll post some more explaining how to build one and how
to extract and purify DNA to run through it. http://www.instructables.com/id/5_minute_DNA_Extraction_in_a...
Want to help? Ping me: mac at diybio.org
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Trifluoroacetic
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I've checked out the website it's very cool keep me updated. When I get time to do more experimenting I will do the same.
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Trifluoroacetic
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I'd be more then happy to help out with the project.
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chief
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DNA-extraction for everyone, very good idea. So one could track all the litle crimes, which happen and are not provable (Who stole the letter from the
postbox ? Who read my private documents? Who sabotaged this or that ? etc.) oneself.
Or: Who let's his dog shit in front of my door all the time ?
Can it be done without too special or expensive chemicals ? I may order a lot of different things, too ... but neither want to get ripped for the
money nor appear in the wrong context in any databases ... ?
Besides: All those private detectives everywhere: One could establish a service for them; ... it would help a lot definately ... !
It definately should not be regulated by any law, since it's probably only lightweight chemistry and a bit of electrophoresis ... so it would be legal
to offer it as a service !
At least the gel-column could be made oneself, then be adjusted via a known standard-substance (2 runs: one with standard, one without; the the
scaling via the standard would be possible) ...
[Edited on 10-12-2008 by chief]
[Edited on 10-12-2008 by chief]
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Trifluoroacetic
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DNA is analyzed through electrophoresis visually by comparing the bands to molecular weight markers that are added to the first column in the gel.
It really that hard to extract DNA and purify it or even do electrophoresis with it. The chemicals that are required in most cases are easily obtained
and quite cheap.
The only real problem that I can see with conducting an Actual DNA analysis is with obtaining and storing the restriction enzymes and PCR enzymes.
They have to be stored at a -20 degrees and are quite expensive. But with that in mind I'd still like to buy some and do some real experimenting.
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mayko
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Extraction of DNA from Drosophilia
Here is the protocol we use at work, when we are sacrificing fruit flies at the altar of reductionism.
Reagents Required
• Bender Buffer
• 5M Potassium Acetate (KoAc)
• Phenol:Chloroform, not acidic
• Chloroform
• Ice cold 70- 85% Ethanol
• Ice cold 95% Ethanol
• Rnase-free H20
Bender Buffer Recipe
0.1M NaCl
0.2M Sucrose
0.1M Tris
0.05M EDTA (MW292.24)
0.5% SDS
pH to 9.1 with NaOH
NOTE: Unless otherwise noted, keep samples on ice. Use tubes that are RNAse and DNAse free and autoclaved pipette tips. Use aliquots of DNA-specific
reagents, rather than general purpose lab reagents.
NOTE: All tube and pipette tips that come into contact with TRIZOL, Phenol:Chloroform, or Chloroform must be disposed of in the Phenol: Chloroform
waste bag (in hood). Work with these in the hood when possible (inhaling any amount isn’t good for you)
0. Collect flies for small prep (1 fly, instructions in brackets [ ] ) or for large prep (12 flies) in 1.7 ml, RNAse free tube. 1 fly = 40uL Bender
Buffer = 20uL 5M KoAc = 40uL phenol = 40uL chloroform = 120uL EtOH
1. Add 480uL of Bender Buffer to the tube. [40uL]
2. Grind the sample down until there are no recognizable fly bits.
3. Incubate at 65C (the sandbath) for 30 min, then let cool down to RT (~5-8 min)
4. Add 4uL of 10mg/mL RNase A [1uL], invert a few times.
5. Incubate at 37C for 15min.
6. Add 480uL 5M KoAc [20uL]. Pipette up and down to mix. Incubate on ice for 10 minutes.
7. Centrifuge at 13000x for 5 minutes.
8. DNA is in the supernatant. Move supernatant to new tube and toss the old one. Add 480uL [60uL] Phenol:Chloroform.. Shake tubes well.
9. Centrifuge at max speed for 10min at RT.
10. DNA is in the top layer. Pipette the top layer into a new tube, being careful not to disturb the interface. If this layer is discolored out
cloudy, repeat steps 8-10.
11. Add 480uL [40uL] Chloroform. Close lids tightly and mix by inverting ~ 15 times.
12. Spin at 15000x for 30 seconds.
13. DNA is in the top layer. Pipette it into a new tube. Add 1000uL [120uL ] of fresh, cold
95% EtOH. Incubate on ice for 10 minutes.
14. Spin at max speed for 15 minutes @ 8C.
15. DNA should have formed a pellet. Remove EtOH (everything but the pellet!) by pipetting or just pouring off. Be careful not to lose the pellet.
16. Add 500uL[100uL] 80% EtOH, spin at 8C for another 5 minutes, then remove EtOH.
17. Spin down tube quickly and remove any excess EtOH with a 200uL pipette.
18. Let pellet air dry for 10 min at RT.
19. optional: If still smells of Ethanol, put on sand bath for 5min at 65C.
20. Elute in 52uL [20uL] EB buffer. Pipette around tube sides to elute not-visible DNA. This step can be wildly variable and depends on downstream
intentions. ddH2O might be favorable.
al-khemie is not a terrorist organization
"Chemicals, chemicals... I need chemicals!" - George Hayduke
"Wubbalubba dub-dub!" - Rick Sanchez
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mayko
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A slight variation: RNA extraction from Drosophila. RNA is a little bit more fragile than its deoxyribo cousin, so a different protocol is required.
Self-assemble your own molecular computers at home!
http://www.technologyreview.com/news/410986/computing-with-r...
Attachment: RNA Isolation from Flies.doc (50kB)
This file has been downloaded 1310 times
al-khemie is not a terrorist organization
"Chemicals, chemicals... I need chemicals!" - George Hayduke
"Wubbalubba dub-dub!" - Rick Sanchez
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confused
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the thing about doing actual gel electrophoresis is that DNA stains are expensive, if you're thinking of using Ethidium Bromide, cleanup and disposal
is a mess. But if im not wrong, you can also use Methalene blue for stains and gentian violet as a loading dye.
The really pricey reagents are PCR enzymes,primers, DNA standard and restriction enzymes
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Little_Ghost_again
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A lot of the above seems way out of date, when I get a chance I will write a detailed post on how to use and make a home PCR and how to sequence DNA
at home, the resolution isnt great but the cost is under £30. If you have a flat bed scanner and a copy of matlab then I can also detail how to get
better resolution, chopping and inserting DNA is not hard.
I have seeen this done time and again here in my dads lab, ok the equipment is different but most can be approximated enough.
If you want a complete chromosome to play around with then go for the fruit fly, really easy to separate and very large for a chromosome.
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amyi
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Quote: Originally posted by Tacho  | Excellent link!!
This is exactly the kind of information I would like to see discussed here.
Any other link like that will be very welcome. |
So which kind of links do you want?
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Laurylamido propyl Betain
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DNA extraction?The moment I saw the word ,I thought of so many fiction films .
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calvin
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gel electrophoresis resolution
Has anyone here actually performed gel electrophoresis using either EtBr or other dyes? If so, what sort of resolution is possible with such
techniques? Most of the images I can find online of band patterns are so poorly defined that it doesn't seem possible to actually sequence more than
maybe a dozen bases this way.
The reason I ask is because our school's computer science club wants to build an automated DNA sequencer based on sanger sequencing but using digital
imaging and OpenCV to read the DNA sequence. Would it be possible to sequence 50bp+ sequences this way?
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NitratedKittens
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| Quote: Originally posted by I am a fish | | Quote: | Originally posted by chemoleo
Lol - I am suggesting nothing  -it's for you to interpret!
This a 'Mad Science' Discussion Board, so I thought it only fair to point out that there are human sources of DNA you can purify. Unfortunately, blood
does indeed contain very little DNA (red blood platelets/erythrocytes dont contain nuclei, hence no DNA), so it is hardly a useful source. Else, you
could of course have one kidney removed and purify DNA from that... probably quite an effort and considerably more painful!
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For women who want to isolate their own DNA and think that surgery is going too far:
Another source of DNA is the soft tissue lining the inside of the cheek. The top layer of this can be scraped away quite easily with a teaspoon.
Alternatively, if you're willing to settle for DNA that is partially yours, you could ask a male relative.
| Quote: |
PS Sorry if I offended anyone ethically/morally/religiously - but trust me there are much worse things you could get offended by  |
That sounds like a challenge to me.
[Edited on 13-2-2004 by I am a fish] |
The best way for a woman to isolate her own DNA in large quantities is to centrifuge her blood then extract from the WBC layer.
Or could the epithelial cells shedded during a period contain enough DNA.
[Edited on 28-12-2016 by NitratedKittens]
Basket of kittens for you ........BOOM
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nighthawk
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sperm DNA extract
How would you extract this DNA? Would a light microscope work to see anything?
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TriiodideFrog
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I suggest using your tongue to dislodge some cheek cells, then spitting your saliva into a test tube.
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TriiodideFrog
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You can also make homemade DNA extraction solution. Fill a beaker with 1/2 cup of water. Slowly add in 2 teaspoons of dish soap and 1/2 teaspoon of
table salt. Gently mix this solution without making bubbles until the salt dissolves. Fill he test tube with saliva 3/4 with the solution. Then,
ice-cold alcohol is added to the solution causes the DNA to precipitate out. The DNA will look like stands of fine silk. You can also try this
experiment with crushed strawberries.
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