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Abromination
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[*] posted on 26-8-2018 at 22:21
Paper Centrifuge


My biology teacher said if I can isolate pure DNA from strawberries, I will get a crap load of extra credit. Now, the DNA you have extracted in the past in high school AP bio was probably not pure. It would look brown, being contaminated with all sorts of cell proteins. To separate the two, we need a centrifuge. We all know jusy how much those cost, so I decided to make my own. After quickly doing some reasearch, I found that Harvard had designed a paper centrifuge for detecting malaria, and it was hand operated. Who remembers the whirligig?

https://m.youtube.com/watch?v=L5ppD07DMKQ

Simple physics.
Now, I want to make my own. Using 2 plastic test tubes, one for the sample and one for balast, I will make a cardboard shell and supports to hold my "ghetto" centrifuge tubes in place. Then I will just copy what they did with the rest of the design and hope I did it right.
I suggest that others try this and would like to hear your thoughts on designs and improvements.
I think this could be a big thing for amateur chemistry.




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[*] posted on 27-8-2018 at 01:24


I have safety concerns:

. the centrifugal force is M.R.W2 ... (M=kg, R=m, W=2.pi.rpm/60)
. the demo showed small capillary tubes = low mass
. you are considering test tubes = high mass

To calculate the speed that an inadequately secured test tube will fly towards you, use this formula;
V = R.W ... (V=m/s)

i.e. use a low mass 'test tube' and
NEVER spin your centrifuge in the plane of your body, and especially eyes.
IF the capillary tubes in that video had broken loose it could have been very bad day.

I do not own a centrifuge but I have seen, and narrowly escaped, flying debris from rotating shafts ... scary.
At the very least, always wear good eye protection EVERY time you spin-up.
This is why 'pro' centrifuges are totally enclosed.
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[*] posted on 27-8-2018 at 02:04


Don't worry Sulaiman.
Mass is very low.

Tape 3 bits of velcro to the card you are spinning, and then stick a circle made of card and velcro over that. It is a great lid, and will stop accidents.

I have made a few of these, and they are ok for getting hematocrit (% of red blood cells in blood: hint, use a single grain of kitchen citric acid to a drop of blood to prevent clotting. Hint 2, don't do hint 1 with anyone elses blood or in front of students)

I find the big problem is that the place the string goes through the card gets torn after a few spins.

Abromination: I think better for amateur chem would be dremelfuge - google it. A little 3d print and easily 10 000g, which will pellet anything larger than a mitochondrion in 10 minutes.

Have fun now!
H.
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[*] posted on 27-8-2018 at 02:11


Further thought:
The DNA you isolated has lots of proteins associated with it.

Perhaps, once you have got the snotty gob of DNA out, you might try to treat it with meat tenderiser or contact lens cleaner (papain/bromelain) to digest the histones and other associated proteins.

There must be a protocol out there.

When you have pure, to persuade your teacher that this is so, perhaps wet ash and measure phosphate: nitrate ratio, which should be doable with water test kits. I would suggest that your control would be a known concentration "spike" of nitrate and phosphate.

So lets say your test kit says your sample has 1mmol of P and 1mmol of N.
You add 1mmol of a phosphate to the sample, ash it and measure again. If your assay and preparation works, you should now measure 2mmol of P.

You can then compare your N : P ratio with the expected value for that DNA and show that your sample is pure, or at least, give a percentage of how pure it is.

Have fun now!
H.


[Edited on 27-8-2018 by Harristotle]

[Edited on 27-8-2018 by Harristotle]

[Edited on 27-8-2018 by Harristotle]
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[*] posted on 27-8-2018 at 02:22


The whole point of a centrifuge it to create large accelerations. The video talks of tens or hundreds of thousand of rpm.

Just calculate the forces on a pair of 10 gram test tubes spinning on a 10 cm diameter card at 1000 revolutions per second before you design something held together with paper and velcro.
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[*] posted on 27-8-2018 at 02:34


Quote: Originally posted by Harristotle  
I find the big problem is that the place the string goes through the card gets torn after a few spins.


As a child I used clothing buttons to make spinners
button.jpg - 23kB
maybe you could glue or tie one (or two, one each side) to the centre of the disk ?
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[*] posted on 27-8-2018 at 02:47


It peaks at 10s of thousands of RPM. It isn't continuous, by any stretch of the imagination.

Buttons look good, but what makes the paper safe/good is its low mass.
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[*] posted on 27-8-2018 at 03:39


No centrifuge required
https://www.youtube.com/watch?v=UDKm9rZbhyI
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[*] posted on 27-8-2018 at 06:04


Could get yourself a professionally made one for 20 bucks if all else fails.
https://www.ebay.com/i/111409028151?chn=ps

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[*] posted on 27-8-2018 at 06:36


Quote: Originally posted by unionised  
No centrifuge required
https://www.youtube.com/watch?v=UDKm9rZbhyI


The blob this guy calls DNA probably contains DNA, but is more than 99% protein junk. If you can get away with this with your teacher, go for it, but otherwise maybe the silica method could be used. A centrifuge could help but I don't think it is necessary.

I'm making this up at the spot, so be warned, but I think it should work:

1. Freeze your strawberries (20 orso, we want to see something in the end) to -20/-300C, also freeze a mortar and pestle, crush the strawberries. Mix and dissolve the now thawed slush in suspension buffer (A), filter out solids, and add binding buffer (B) (3 volume parts A plus strawberry and 1 volume B).
2. throw in an amount of silica and stir for about half an hour or so. Naturally occurring RNAses will take care of the present RNA. Filter out the silica (now has DNA bound), and wash with washing buffer (C).
3. Re-suspend the silica in elution buffer (D), stir for about half an hour or so. Filter of the silica, the solution now has mostly only DNA. Rinse the silica with solution D. Add absolute ethanol or isopropanol to a final volume of 70% alcohol and cool to -20/-300C for about an hour. **

The DNA, if it bound to the silica (I think this is the crucial step, the rest is quite foolproof) will now be visible as a small white thingy floating around or stuck to the bottom if centrifuged.

I don't have time now, but tomorrow I will have a look for the components of the different solutions (A-D), but they are quite easy to find online if you are in a hurry.

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[*] posted on 27-8-2018 at 09:34


The current version of the Paperfuge is published in Nature BME which I can only get through SciHub, meaning no SI. But you can probably find a lot of build details and other information in the bioarxiv preprint. There's some other good discussion on DIY centrifuges in the surrounding thread here:

https://www.sciencemadness.org/whisper/viewthread.php?tid=14...

there's also some information on DNA extraction in this thread:

https://www.sciencemadness.org/talk/viewthread.php?tid=1496&...


Attachment: Hand-powered ultralow-cost paper centrifuge.pdf (6.1MB)
This file has been downloaded 236 times




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[*] posted on 27-8-2018 at 09:36


This is the protocol that I have used in the past to isolate DNA from mouse tail tips. No silica is required. The DNA is of relatively high purity but does have some RNA (if needed, RNase can be added to remove this).

1) To the tail tip, add 100 µL TENS buffer (pH 10.5, 10 mM Tris, 1 mM EDTA, 150 mM NaCl, 0.5% SDS) with proteinase K. Incubate at 60 °C until the tip dissolves (usually about 30 minutes).
2) Cool to rt and add 33 µL sat. NaCl. Mix and spin at 14,000 g for 1 minute (note: this can also be a lower speed for longer time)
3) Transfer supernatant to a new tube and add 200 µL cold isopropanol. Mix and spin at 14,000 g for 2 minutes. A pellet of DNA will form.
4) Discard the supernatant and resuspend the pellet in ~100 µL cold 70% ethanol (precise amount doesn't matter). Spin at 14,000 g for 1 minute, then remove the supernatant.
5) Let the pellet air dry, then dissolve it in 100 uL pH 8.0 10 mM Tris, 1 mM EDTA buffer. The DNA concentration can now be measured.


Edit: The most difficult step for the centrifuge is the initial pelleting of the DNA. This can be done at as low as 3000 g (I have done this on a larger scale, when high g-forces are a problem due to limited container strength). However, at 3000 g it takes a pretty long time, up to an hour.

[Edited on 8-27-2018 by Metacelsus]




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[*] posted on 27-8-2018 at 11:39


Thanks, everyone. I am aware of all of the proteins mixed in with the DNA and thats what I need to remove. Ill consider all of these and think about them more when I have the time. The test tubes that Im using are not what you are probably thinking. They are only a bit larger than centrifuge tubes.

IMG_3243.JPG - 1.9MB

Can I use this method of extraction (used in my class) and centrifuge it to remove the proteins?

https://m.youtube.com/watch?v=

[Edited on 27-8-18 by Abromination]

[Edited on 28-8-18 by Abromination]




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[*] posted on 27-8-2018 at 23:49


@ Abromination; your link is incomplete

Quote: Originally posted by Metacelsus  
This is the protocol that I have used in the past to isolate DNA from mouse tail tips. No silica is required.
[Edited on 8-27-2018 by Metacelsus]


The reason why I posted my silica protocol is because it avoids the need for very high rpm centrifuges. With the silica method you don't reallyneed a centrifuge, or if it is used to make filter separations more efficient (recommended) you only need short period low g spin cycles (doable with the paperfuge).

I do see now how a different step 1 would more efficient in getting rid of proteins with basic chemicals.

New step 1; Crush the strawberries in buffer A (freezing isn't necessary), leave at room temperature for half an hour (let the natural RNAses do their work), add buffer A2 (denatures proteins) and mix untill one uniform snotty gel, add Buffer A3 (crashes out proteins but not DNA) and mix untill uniform liquid again. Filter (or centrifuge low g with paperfuge). Mix supernatant with silica and proceed with step 2.
-- Doing it this way avoids the need for proteinase K. --

Buffer A; 25 mM Tris, 10 mM EDTA (pH 8.0)
Buffer A2; 0.2 M NaOH, 1% SDS (1% dish washing detergent should work as well).
Buffer A3; 5 M potassium acetate (pH 5.5)
Buffer B is not necessary anymore
Buffer C; 20 mM NaCl, 2 mM Tris (pH 7.5), 70% ethanol/isopropanol
Buffer D; 10 mM Tris-HCl (pH 8.5)

So you need silica, Tris, EDTA, NaOH, detergent, K-acetate, alcohol, NaCl and a low g centrifuge if available. And a pH-meter to set the buffers.

You can add a pinch of methylene blue (MB) to buffer A2 to check if buffer A3 is doing its job (become colourless).


EDIT
Volumes needed:
- 3 parts (volume) buffer A for 1 part strawberry.
- 1 part buffer A2 on 1 part strawberry/buffer A mix
- 3.5 parts A3 on 5 parts strawberry/A/A2 (or untill colorless when MB is used) (please check these proportions, it should become around pH 6 / 6.5, you can neglect the strawberries in these calculations).
- Wash buffer two times (separate in two washings) the volume of the silica
- Elution buffer the same volume as the silica
- absolute alcohol as much as needed to reach 70% final volume


[Edited on 28-8-2018 by Tsjerk]

[Edited on 28-8-2018 by Tsjerk]

[Edited on 28-8-2018 by Tsjerk]
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[*] posted on 28-8-2018 at 04:35


How selective are the sorts of proteases used in home brewing??
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[*] posted on 28-8-2018 at 07:34


Quote: Originally posted by unionised  
How selective are the sorts of proteases used in home brewing??


What are their names? Do you have a link to a product?
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[*] posted on 28-8-2018 at 15:07


With brewing proteases, I would be worried about contamination with nucleases. I doubt the manufacturers of brewing-grade proteases take special care to remove these. Still, it can't hurt to try.

Another possible method for removing protein is phenol-chloroform extraction. See: https://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extr...

[Edited on 8-28-2018 by Metacelsus]




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[*] posted on 28-8-2018 at 17:26


Quote: Originally posted by Tsjerk  
@ Abromination; your link is incomplete

Quote: Originally posted by Metacelsus  
This is the protocol that I have used in the past to isolate DNA from mouse tail tips. No silica is required.
[Edited on 8-27-2018 by Metacelsus]


The reason why I posted my silica protocol is because it avoids the need for very high rpm centrifuges. With the silica method you don't reallyneed a centrifuge, or if it is used to make filter separations more efficient (recommended) you only need short period low g spin cycles (doable with the paperfuge).

I do see now how a different step 1 would more efficient in getting rid of proteins with basic chemicals.

New step 1; Crush the strawberries in buffer A (freezing isn't necessary), leave at room temperature for half an hour (let the natural RNAses do their work), add buffer A2 (denatures proteins) and mix untill one uniform snotty gel, add Buffer A3 (crashes out proteins but not DNA) and mix untill uniform liquid again. Filter (or centrifuge low g with paperfuge). Mix supernatant with silica and proceed with step 2.
-- Doing it this way avoids the need for proteinase K. --

Buffer A; 25 mM Tris, 10 mM EDTA (pH 8.0)
Buffer A2; 0.2 M NaOH, 1% SDS (1% dish washing detergent should work as well).
Buffer A3; 5 M potassium acetate (pH 5.5)
Buffer B is not necessary anymore
Buffer C; 20 mM NaCl, 2 mM Tris (pH 7.5), 70% ethanol/isopropanol
Buffer D; 10 mM Tris-HCl (pH 8.5)

So you need silica, Tris, EDTA, NaOH, detergent, K-acetate, alcohol, NaCl and a low g centrifuge if available. And a pH-meter to set the buffers.

You can add a pinch of methylene blue (MB) to buffer A2 to check if buffer A3 is doing its job (become colourless).


EDIT
Volumes needed:
- 3 parts (volume) buffer A for 1 part strawberry.
- 1 part buffer A2 on 1 part strawberry/buffer A mix
- 3.5 parts A3 on 5 parts strawberry/A/A2 (or untill colorless when MB is used) (please check these proportions, it should become around pH 6 / 6.5, you can neglect the strawberries in these calculations).
- Wash buffer two times (separate in two washings) the volume of the silica
- Elution buffer the same volume as the silica
- absolute alcohol as much as needed to reach 70% final volume



[Edited on 28-8-2018 by Tsjerk]

[Edited on 28-8-2018 by Tsjerk]

[Edited on 28-8-2018 by Tsjerk]


I can not get my hands on Tris, EDTA or HCl. I actually probably could get HCl but what I get is at my schools discretion. Any better way?




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[*] posted on 28-8-2018 at 22:33


Then phenol /chloroform is probably also it if the question right? You could do a phenol chloroform followed by a alcohol precipitation.
I could imaging you use hydrogenphosphate / dihydrogenphosphate instead of Tris, but I don't know if I can find a reference for that, I will have a look today.

If you use a phosphate buffer and measure the salts carefully you don't need a strong acid anymore. Use acetic acid to acidify the K-acetate, Na-acetate is also fine.

EDTA is there to catch divalent cations that are essential for nucleases to work (except RNAse), if you keep everything on ice all the time it should be fine without.

Edit: check if the phosphate doesn't precipitate with the alcohol beforehand by using the protocol without strawberries, there shouldn't be any precipitate anywhere down the line.


[Edited on 29-8-2018 by Tsjerk]
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[*] posted on 29-8-2018 at 21:36


Quote: Originally posted by Tsjerk  
Then phenol /chloroform is probably also it if the question right? You could do a phenol chloroform followed by a alcohol precipitation.
I could imaging you use hydrogenphosphate / dihydrogenphosphate instead of Tris, but I don't know if I can find a reference for that, I will have a look today.

If you use a phosphate buffer and measure the salts carefully you don't need a strong acid anymore. Use acetic acid to acidify the K-acetate, Na-acetate is also fine.

EDTA is there to catch divalent cations that are essential for nucleases to work (except RNAse), if you keep everything on ice all the time it should be fine without.

Edit: check if the phosphate doesn't precipitate with the alcohol beforehand by using the protocol without strawberries, there shouldn't be any precipitate anywhere down the line.


[Edited on 29-8-2018 by Tsjerk]

Thanks. I can probably do this as all I really have to do (this winter of course) is stick my stuff outside and it should freeze fairly fast. Infact I probably could just work outside. So that takes care of the EDTA problem. I can use disodium phosphate for my Tris substitute (as was pointed out im on a REALLY thin budget of like 30 USD or something) and thats another solved problem there. Phenol and chloroform are indeed out of the question *sigh*
I don't have my own and the school is not going to let me use chloroform. Maybe phenol but I really doubt it.
But, me being terrible at biochemistry (I'm an inorganic guy) are you saying that chloroform/phenol is nessesary for this method? If not I am home free.




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[*] posted on 29-8-2018 at 21:49


No, phenol/chloroform is one technique, the Tris(or phosphate) /NaOH/detergent is another. So you don't need chloroform or phenol.

I see my protocol is a bit scattered over multiple posts now, if you want to use it you could write it down and post it here so I/we could have a look at it.
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[*] posted on 6-9-2018 at 11:40


Quote: Originally posted by Tsjerk  
No, phenol/chloroform is one technique, the Tris(or phosphate) /NaOH/detergent is another. So you don't need chloroform or phenol.

I see my protocol is a bit scattered over multiple posts now, if you want to use it you could write it down and post it here so I/we could have a look at it.


Thats what I thought. I have it all written down in my notebook, but have been a bit busy lately. I will post it when I have the chance.




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[*] posted on 7-9-2018 at 11:18


Can someone please tell me why OP doesnt just put a drill in a bench vise and use it to centrifuge a test tube that way?
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[*] posted on 7-9-2018 at 13:45


Because the OP doesn't want to distribute his sample in a 360 degrees manner
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[*] posted on 11-9-2018 at 07:39


If all else fails, there's always the FidgetFuge:

https://twitter.com/ABRC_News/status/913870761810694144




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