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Author: Subject: Extraction of DNA
Trifluoroacetic
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wink.gif posted on 6-8-2008 at 19:42
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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[*] posted on 7-8-2008 at 17:24
DNAExtraction




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[*] posted on 7-8-2008 at 17:26
DNAExtraction


Here is a test run of my homemade tracking Dye.

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[*] posted on 12-11-2008 at 15:34


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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[*] posted on 9-12-2008 at 18:28


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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[*] posted on 9-12-2008 at 18:29


I'd be more then happy to help out with the project.
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[*] posted on 10-12-2008 at 10:20


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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[*] posted on 10-12-2008 at 17:51


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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[*] posted on 13-10-2013 at 09:45
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.






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[*] posted on 16-10-2013 at 10:31


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)
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[*] posted on 16-10-2013 at 10:54


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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[*] posted on 21-10-2014 at 12:13


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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