DNA Extraction and Restriction Enzyme Protocol

I promised I would have something posted for you by the end of the week, so here it is, the full protocol I used in my experiment, as well as directions on how to make any substances/solutions I think you shouldn't be expected to understand. My eventual goal is to annotate this with my reasoning for each step, as well as advice on how to complete some of the more vague steps, but unfortunately I have come down with a monster lung infection and was hard pressed to get this posted. I hope that this coming advice, tips and tricks article will help those new to science, or even just new to many step protocols, and will more than anything help you become a person that can plan something like this (or much better!) on your own. Since this was put together for use by a high school teacher, I'd like to explain that the licensing on this site is creative commons, which means you are free to distribute this as long as you don't make money off of it, and attribute it to me (throw my name in .2 font on the bottom of the last page, not worried about that for teachers). I hope someone will find a use for this - if you do, leave a comment. :) Without further blathering on my part, here it is:

Protocol for the Extraction, Restriction Digestion and Electrophoresis Analysis of Human cDNA

Collect cell sample by swishing with gatorade for 1 minute.

Withdraw .7mL of cell solution and transfer via transfer pipette to a 1.5mL micro centrifuge tube.

Transfer 125uL of lysis solution (clear dish washing soap) to the 1.5mL micro centrifuge tube with a new transfer pipette.

Invert micro centrifuge tube for 1 minute, and allow to sit for another minute.

Transfer 250uL of papain solution (see solution list) to the micro centrifuge tube via transfer pipette.

Invert sample for one minute.

Heat micro centrifuge tube to 65C for 10 minutes.

Centrifuge tube to pelletize cell waste and any other adulterants (mainly some of the heavier ingredients in the soap).

Collect .5mL supernatant and transfer via transfer pipette to a new micro centrifuge tube.

Transfer 1mL of 0C ethanol to micro centrifuge tube and allow to sit for 5 minutes at ~20C.

Centrifuge until visible pellet forms.

Remove supernatant by transfer pipette (and micropipette) if necessary, not disturbing the centrifuge pellet.

Allow the pellet to air dry in the tube for several minutes.

Resuspend pellet (DNA) with 43uL dH2O.

Add 5uL of NEBuffer 4.

Allow pellet to fully dissolve before proceeding, using 65C heat and vortexing (or spinning arm in a circle) if necessary.

Transfer 1uL of 10,000U/mL AluI restriction enzyme via micropipette.

Transfer 1uL of 20,000U/mL EcoR1 restriction enzyme via micropipette.

Vortex (or spinning arm mix) to assure a homogenous mixture.

Heat micro centrifuge tube to 37C for 2 hours.

Heat micro centrifuge tube to 65C for 20 minutes.

Transfer 10uL of Bromophenol Blue gel loading dye (6x) to the micro centrifuge tube via micropipette.

In gel electrophoresis chamber, insert gel and fill with 1x TBE until the gel is completely covered.

Load 60uL loading solution into gel electrophoresis slab wells.

Run at up to 63 volts until the bromophenol blue dye has migrated to the edge of the gel slab.

Remove gel slab and put into a dye chamber.

Cover gel with .003% Methylene Blue and allow to sit until DNA bands become clear.

5x TBE Buffer (1L):

53g Tris base

27.5g Boric acid

20mL .5M sodium EDTA (pH 8.0)

dH20 to 1L

Methylene Blue .2% stock solution (100mL):

.2g Methylene Blue trihydrate

dH20 to 100mL

Methylene Blue .002% staining solution (500mL):

31.25mL .2% Methylene Blue stock solution

468.75mL dH2O

1x TBE Buffer (1L):

200mL 5x TBE Buffer

800mL dH2O

Agar(ose) gel 1%:

1g agar(ose)

1x TBE to 100mL

Heat in microwave in 10 second intervals, being careful not to boil (emulsion is impossible to resolve and will result in poor electrophoresis), stirring gently (once agar(ose) starts to melt solution aerates very easily).

Pour ~50mL for each Ward's gel electrophoresis form, or however much is prescribed by your electrophoresis chamber.

Saturated papain solution:

10mg/ml of dH2O – prepare amount desired, heat to aid in dissolving (a small amount of powder won't dissolve, I suspect this is the clumping agent/other non-papain ingredients which aren't water soluble – filter it if you really feel it's necessary)

Bromophenol Blue Loading Buffer 6x: obtain from New England Biolabs (free to high schools), or find a similar loading buffer recipe online and make yourself.

AluI and EcoR1 Restriction Enzymes: order from New England Biolabs (free to high schools, just call their support number and ask about their discount program).

Looking Back and Looking Ahead

Last week was stressful and incredibly exciting for me. First off, I completed the experiment I have been preparing for a local high school, and saw it yield very respectable results (considering the constraints we faced). Secondly, I received my OpenPCR kit.

The school experiment was my first real foray into the DIYBio world, and showed me that with enough determination it is possible to do things that at first glance would seem impossible under the circumstances, as well as showing me that it is possible to engage lay-people in advanced science if you use simple concise language and allow them to actually participate in the science and see tangible results. I'll be writing a technical review of our methods, where we saw success, and where we had short-comings over the next week but can say that overall I think the project was a success, and that we will be continuing to develop protocols and experimental frameworks to show the essentials of biotech to lay-people, and allow scientists with limited resources to perform useful work within the area of genetics.

I also received my OpenPCR kit this friday, the same day I finished my first experiment above. Once I got it alone in my lab/bedroom that night, I locked the doors, turned on every light I had (all the better to see you, my pretty :P), and spent about four and a half hours assembling the machine: peeling backing off of insulation, obsessively lining up heating elements, screwing many screws and being very careful of a certain temperature probe wire which I was told was so fragile that breathing on it would break it and ruin the lives of my family for generations to come (:P).

But, with surprisingly little crying or grunting I got the machine together and started running it through it's paces. I very quickly was flabbergasted as this machine I put together (convinced the entire time that it was never going to turn on, or would crack the heating elements the first time I turned it on) started up like a well oiled machine, and proceeded to perform BETTER than its technical specifications. I found that it could achieve a low temperature of 0.3 degrees celsius in an ambient temperature of 24 degrees. It heated up at a rate of 1 degree celsius, but only at the high and low point of its temperature range - it performed better than 1C/second in the mid range of its temperature, where both the annealing and extending temperatures are found. So, shocked, I set the machine on my lab table (improvised) and went to bed to go to work in the morning.

Now I sit here on Sunday afternoon relating all of this to you, in a sort of awe of how far I've come in the last several months, and looking ahead at what I plan to do in the next six months and, further on, in the coming years. It reminds me of that moment of a rollercoaster when the interminable clacking from being pulled up the first hill stops, but before the rollers start rushing from the downward slide into the rest of the coaster. That is this moment I think, and I can confidently give you the advice that when you find yourself in this moment, take a second to check your calculations - and then lift your arms up and prepare for the ride of a lifetime.

I'm being lazy today, resting, so I'm not going to post any real science content, but wanted to describe this feeling of having gotten over the first hill and looking out over the others that will be mounted by sheer inertia. I'll fully write up my first school experiment over the next week, and will provide at least a sketch of a plan for my work over the next six months, but for now I am going to track down a glass of raspberry tea, go sit on my porch, and enjoy the unseasonably warm weather for a moment. :)

News From the Frontlines

You have to see the view from here for yourself to really believe it, but this might do it justice if you squint and try to see the hopes and dreams of the next generation floating around in the background. This is where I've posted myself over the last weeks, working on a genetics demonstration for a high school in my area. We live in a semi-rural community, so money is tight, but the kids are bright and the teachers are more than willing to give a man who calls himself a "Biohacker" a chance. Insert this biohacker into the mix, and you get a plan to do a human DNA extraction, run a digestion reaction based on the Alu I and Eco R1 repeat sequences, and do an electrophoresis analysis on the products of said digestion. All on a shoe string budget and the charity of the good folks at New England Biolabs, who are good enough to donate any product they sell to high schools in need.

I've told you all of this before, and given a few technical details as well, but the project is finally coming to fruition, and I feel the need to kick back and tell a yarn for a moment.

Today we received the final tool we were waiting for, a micropipette from quasar instruments (cheap but good pipettes). Also, today we ran the second experiment in three days: a simple graduated cylinder extraction of human cDNA (see pictures on a previous post). Previously, the kids had done a banana DNA extraction to get the basic idea of the protocol, this time we introduced swishing to collect cheek cells and papain as a protease. (sorry bout the lack of in action photos, youngins can't sign over their photo rights) By next week we'll be running the full protocol including centrifuges, micropipettes, restriction enzymes and electrophoresis with methylene blue staining.

For me today wasn't just a performance, it was a day of preparing for DNA war, and my main goal was to figure out what percent concentration of agar (that's right, agar, not agarose -though we use TBE, not McGuyver buffers like other protocols) is right for our electrophoresis analysis. Note of experience: don't put 50 mLs of agar mixture in the microwave for 30 seconds, then another 20... It results a panic when you think you ruined the teacher you're working with's meal cooker, only to discover that a previous biology teacher had already done this to the librarian with burnt yeast and caused him to turn over his microwave to the science department for chemical cooking.


In the end, the 1% agar solution won the war and stole my heart, contrary to literature saying 2% was called for.

I know this is a bit of a strange post for me, and that most of you are wondering where the protocols are at. I'll post a very detailed technical writeup once this demonstration is finished (should be next week at the end of the week), until then be satisfied with this picture memoir. I wanted to post the pictures tonight, but wanted to relax and forget about technical details for a minute.
It's back to the lab again tomorrow... I hope you get to learn how fun it is to say that sometime.



***Blogger sell-out note: None of these companies are sponsoring me or anything, they have been good sources of equipment, so I advertise and link them here for your benefit and without their knowledge***

I've done something silly...

...And it involves large sums of money. Last night I put in an order for an OpenPCR machine from openpcr.com. Their unit costs $639 with domestic shipping included, comes as a build it yourself kit (minus having to solder boards), and promises to change the world as long as people actually find out about it.

I know you may be thinking, has our blog author fallen off the one true faith? Given up neuroscience? The answer is a distinct "Not really." In the realm of neuroscience I've found myself asking more and more how does a certain set of receptor signals change how the neuron works forever more? Inevitably, once you follow a few signal transductions -and an enzyme cascade for many more steps-, this leads to genetic expression alterations. As I've said before, I feel that the multi-disciplinary part of neuroscience is lacking, and so I'm becoming a geneticist to become a better neuroscientist.

Over the next few months I will be continuing my neuroscience pursuits (which I'll catch you up with in a more detailed post later), but will also be pursuing genetics (and thus the heart of diybio) with new vigor. I'll probably be using this blog as a record of, and sounding board for, those experiments with genetics, so don't be shocked to see a lot more pure genetics material on this blog in the near future (though I'll be sure to tie it back into neuroscience wherever possible).

I hope you'll tolerate my seemingly bifurcating pursuits and trust that I really do have an end goal in mind, and if not an end goal then a plan which will shine some much needed light on just about every bio topic that exists today. Look forward to a real update soon, I've been too busy working on my various projects to post about them, but couldn't contain my need to sing "The Hills Are Alive, With The Sound Of OpenPCR" from the mountain tops.

Where I've Been and Where I'm Going

I promise there's a reason I haven't been posting to this blog as of late, and it isn't at all related to an excuse about my cat eating my blog drafts.

First, on the high school lab demonstration front, I've been stalled waiting for the chemicals to do the electrophoresis portion of the lab. We recently received the enzymes we needed from New England Biolabs, but haven't received the buffer chemicals to do any sort of qualitative analysis of the effects of the enzymes on our DNA samples. So, there hasn't been any work to be posted.

Second, on the front of neuroscience, I've recently decided that I am going to need a whole slurry of advanced math knowledge that I currently don't posses. As I have said before, my college math exposure is severely limited, and I've been acquiring the skills necessary to do advanced calculus and work with differential equations. To that end, I've been running through the Khan Academy programs on calculus and differential equations, as well as several resources I've found on the web, including the MIT OpenCourseware 18.01,18.02 and 18.03 video lectures. The "Paul's Math Notes" resource has also proved invaluable, available at http://tutorial.math.lamar.edu/Classes/. Note that anything I state incorrectly about math in the future should in no way reflect the quality of material at any of these sites, only my inability to absorb the information in the small amount of time I have allotted myself.

Also, I've been doing research on the structure of the neocortex in mammals, and our understanding of the processes their in. I've especially been focusing on the enzyme cascades involved in g linked protein receptors, as so far I've seen no evidence that these massive enzyme reactions are taken into account in the mathematical models of the brain I've seen so far (though I've only been looking at single neuron models, though most higher level models seem to be simplifications of the single cell models).

Over all, it seems that there is a lack of communication across the intra-discipline facets of neuroscience. The biologists aren't talking the physiologists, and everybody has their own mathematician, while the information theory guys sit in a corner and play with idealized circuits that no more approximate actual neurons than a light switch approximates a modern cpu. Maybe I'm wrong, but it seems to me like a good deal of the knowledge needed to understand the brain exists, but has never been combined (with the synthesis of new relations and destruction of dead end supposition that that would imply).

I find myself wondering why this hasn't happened, and almost am unable to believe that it isn't some gross oversight of recent research on my part. But if it isn't an oversight on my part, I wonder why this coming together of the various researchers in a self proclaimed inter-discipline science has never seemed to happen.

For example, I have an affinity for the serotonin g linked protein receptor cascades. I think that the interaction of the raphe nucleus (the main serotonin producer in the brain), which connects to almost every other part of the brain, including the neocortex, could be a very interesting site to study the down stream effects of these enzyme cascades on overall neuron firing. Yet nobody seems to have talked about it except in reference to sleep cycles.

The same goes for every other thought I have about the brain as a whole. Everybody seems to be doing studies of these small problems, creating points of understanding, but nobody seems to be drawing the thick connecting lines that are going to outline the actual functioning of the brain as a whole. Perhaps this is a sophomoric line of reasoning, and maybe it is that they want to be certain of their conclusions before they integrate that understanding into the larger image of the brain, but it seems to me like there has to be a connecting of assertions at some point, and i think connecting these small studies would inform them just as putting a part of a machine into a simulation of the machine and having the machine either work or not work would inform the process of creating the part.

It doesn't help that there aren't many killer applications of a whole brain understanding, at least that the few corporations involved in brain research are willing to see. Most applications that involve making money seem to be centered around developing an almost autistic understanding of a single process in the brain (memory for alzheimer's and dementia research, mood for the psycho-pharmaceutical companies, etc.), and university researchers seem almost afraid of stating any conclusions about the brain in general for fear of being wrong.

So, I've been doing a lot of thought experiments and studying, and not a lot of anything that you would want to read about on a blog. Most of the time I find myself wandering around in a daze, puzzling over the implications of a certain assertion about the functioning of the brain, trying to picture giant logic charts in my head. When I'm not doing that  either have my head shoved hermetically into a piece of graph paper doing a problem, or reading/watching on the computer until my eyes or head hurt, whichever comes first. It doesn't help that my hours at my day job have recently been cut back, so my ability to buy equipment has been decidedly hampered.

But I promise you this, I am still most definitely interested and active in the topics of this blog, I'm not dead, and haven't abandoned this site as so many people do with websites after a month or two. I've just found myself without anything to publish, or so I thought until I look above this cursor and see a huge screed laid out (though arguably more of it seems to be about what I haven't figured out than what I have figured out, but there's something about wisdom that comes to mind when I see that). I'll continue updating as I come upon things worth blogging about, but can't promise there will be a heaping of blog posts for whoever reads this every week, or every month for that matter. Put me on your update list and keep an eye out, but holding your breath may prove unhealthy.

What Have I Been Doing?

Just what Have I been doing lately? I certainly wasn't posting on this blog, as is evident from the time stamp on my last post. I've been working on the high school science class demonstration I outlined in my last post, and doing a LOT of reading on neuron imaging, current injection and computational models (both theoretical and practical).

On the front of science demonstrations, I've made a lot of progress as far as pinning down protocols, and now am mainly just waiting for supplies to arrive so I can start testing the techniques and protocols I've conglomerated from resources online and the helpful posts of fellow biohackers on the Diybio google group. I've posted a first tested draft of a human DNA extraction protocol on this blog for the world to start perusing (if the world ever manages to find my blog), and it is workable if not incredibly coarse and still slightly unquantified (ran out of time to test the optimal amount of meat tenderizer protease for the mixture, and will probably need to see results in electrophoresis before I can really determine what ratio and conditions are optimal or even make a difference).

I spent five hours yesterday in the high school lab working on different scales of extraction and trying to coax the DNA I extracted into "spooling" onto a bamboo shoot (as is talked about here, here and just about everywhere else that uses a protocol like the one mine is based off). My conclusion is that either my stirring/enzyme use has somehow not yielded the structural form of DNA that their experiment yields (shearing or DNA structural proteins still coiling the DNA), or that human and animal DNA behaves differently at a macro level. My specialty isn't genetics so I consulted the diybio group, and their answer is "we haven't ever tried 'spooling' DNA". My conclusion is that spooling human DNA most likely isn't possible, and am going to use a centrifuge to pelletize the DNA im trying to extract.

So, yes, I bought my first piece of lab equipment yesterday. A Dremel 300 from a big box store for $49 US, and a Dremelfuge Rotor designed by Cathal Garvey and sold by Shapeways for a total of $58 US with shipping to the USA (not sure where it's coming from). It should arrive by 11/9. It promises to be a good investment, being able to develop (relatively safely; disclaimer: I'm not responsible if you get shot in the leg or eye by an errant centrifuge tube) just over 50,000Gs worth of force. That's as good as a high grade commercial centrifuge I'm told, and I'm excited to see how it works, and will post pictures, text and maybe even a video of it when it comes in.

In the realm of neuroscience I've been splitting my time between continuing to read about research methods in the realm of neuron imaging and current injection (reading what a neuron does, and making it do what I want it to do respectively), and the mathematical models that exist to describe both a single neuron and the computational nature of many neurons acting in concert.

In the realm of neuron imaging I have gone from reading about recording electrodes full circle back to the realm of fluorescence imaging techniques, this time in reference to voltage sensitive dye techniques, which are less complicated than the voltage sensitive proteins I had read about and possible mentioned earlier in the course of this blog.

Also, I'm trying to pin down and wrap my head around the nature of and the specifications of an electrode capable of injecting a current into a single neuron along the lines of those used in the Dynamic Gap technique, which I have been reading about quite enthusiastically. It sounds like the perfect technique for refining and modifying the single neuron model that I think badly needs attention.

And what better segue could I have asked for to talk about computational neuroscience, aka the art and math of figuring out what these neurons we're observing are doing to information? I've been mostly focused on the single neuron models such as Intercept-Fire, the Hodgekin-Huxley model, and the stochastic models derived from the H-H model. In order to look at system level models I'm going to have to have a good grasp on these first. I'm most definitely only doing research at this time, and to be honest am struggling with the math involved, as my last Calculus class was in my senior year of high school, and even then we didn't get very far in the book. So, right now I'm wrestling with the single neuron model, trying to work my way up, fighting on another front with the math involved with any of this. The single greatest resource I've found so far is a computational neuroscience textbook that I randomly found posted on a website in the .ch domain. I haven't had the time to figure out if it's a legally published copy or not, but it has most definitely been a god send for this scientist.

I apologize for the lack of links in the neuroscience part of this post, and will do a link update in the next few days, but I'm too lazy on my last day off to track down all the articles I've been reading. I did at least bother to find the link to the neuroscience textbook I've been reading, as it's sitting in the tab next to this one on my browser. You'll be hearing a lot more from me, and possibly even seeing me, in the coming weeks, as I ramp up my efforts on this science demo, and continue working on my neuroscience pursuits. Now:


Stand Back, I'm Going to Try Science.

Protocol for the Extraction of Human Genomic DNA

Here is a first usable and tested draft of my protocol for human genomic DNA extraction. It isn't meant to produce lab grade products that would be suitable for PCR, but should be pure enough for analysis by means of a basic gel electrophoresis setup (not tested yet), and was created for a high school science demonstration. I've tried to include some of the basic considerations for doing this project on larger or smaller scales (I've tested between 20 mL and 200 mL cell solution scales), and at this time have excluded the use of a centrifuge step (which will be included in a different version once I get my centrifuge and have time to test the rough draft protocol I have prepared for it). Hopefully this is a little clearer than some of the "MacGuyver" protocols out there, and has been explicitly tested on extracting human DNA.

Human Genomic DNA Extraction:

1. Rinse mouth for ~1 minute using a solution of Gatorade, for large sample sizes you can rinse multiple times.

2. Spit solution into a straight walled beaker of suitable size (see further steps to determine size needed).

3. Measure out and add a quantity of detergent in a ratio of 3 mL detergent/20 mL of cell solution.

4. Stir the solution by a method appropriate for the volume of materials used (for single mL volumes in a microcentrifuge tube, invert; for a 1L beaker, stir with a stirring rod).

5. Add an undetermined as of yet "pinch" of meat tenderizer to the solution. (to be quantified in version 2)

6. Stir again until the meat tenderizer is completely in solution and allow ~5 minutes for it to do it's work.

7. Carefully pour the completed solution into a test tube, graduated cylinder, or other container that will allow a low area of surface at the top of the solution in comparison to its volume.

8. Carefully pour 2x the volume of the cell solution of cold (0 degrees C) alcohol (70%> isopropyl alcohol or lab grade ethyl alcohol) into the cylinder, pouring down the side so as not to create an emulsion at the interface between the two separated layer of solutions.

9. Wait for a time while the DNA (and pollutants such as RNA, etc) precipitate at the interface between the alcohol and the cell/detergent solution.

10. For large volume extractions (such as a 200 mL cell solution and 400 mL of alcohol), it can be helpful to stir the alcohol layer such that it agitates the lower layer into a sort of inverse vortex, allowing more of the cell solution to interface with the alcohol over a shorter period of time, while still mostly preserving the division of the layers.

11. Do what you will with the DNA present. Over a period of time (varies depending on volume from 10 minutes-hours) the DNA will settle onto the bottom of the alcohol layer for easier siphoning off to do further studies, or it can be held there for an undefined period of time as something that simply looks cool (will stay intact for at least a day).

The pertinent ratios of chemicals here is the 3 mL detergent/20 mL solution (for consumer grade detergents) which seems to work well for lysing the cells while not creating to much of a mess in the way of bubbles (which make your interface later in the experiment less defined). Also, the ratio of 2x the volume of cell solution in alcohol seems to facilitate visual inspection of the DNA, as well as providing room for error in pipetting off a DNA sample.

Here's a link(Picasa) to a gallery of a few pictures from my smart phone of the result of this extraction being used on a 200 mL cell solution.

I know this isn't by far a perfect protocol, and there are still a lot of unquantified variables and amounts, but that should change over the coming few weeks.