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. :)

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.

Class Room Genetics, Here I Come!

I posted a few days ago that I was going in to propose that I could help a local high school science teacher run an interactive genetics lab for her students during their upcoming genetics unit. I talked to her for several hours that day, having abandoned my original proposal of a strawberry DNA extraction for a human cheek cell DNA extraction followed by imaging of the student's samples with agarose gel electrophoresis.

This has been modified during the course of my research, and with the invaluable help of the contributors to the Diybio mailing list, which you all should most definitely join. Originally I was ignorantly going to try to run the electrophoresis on unbroken genomic DNA. What I didn't realize was that human chromosomes were entirely too long to be resolved by a gel except as a smear at the top of the gel where it gets stuck in the pores of the agarose.

Thanks to several online collaborators I've been able to figure out the specifics of the electrophoresis process (details of which are surprisingly vague online and in journal articles except for specific protocols, information which is pretty much useless except for the most basic information if you are running a different experiment), and more importantly, how to make a gel run which will have meaningful results that wont end with a smear of clumped and stuck DNA

I'll be publishing several articles here about what I've learned as soon as I get time. Right now I'm too busy doing the actual work of setting up the demo and getting organized to produce a digested record of what I've learned. In the mean time feel free to check out my threads on the Diybio mailing list (google groups) for a day to day reference and to see the process by which I got the information, and the other researchers who have helped me.

Onward and Upward, Challenges and Chances

Well guys, I've done a lot more research and have been talking (and asking copious questions) on the diybio mailing list(google groups). It looks like I have my work cut out for me, and I'm going to have to study a good deal, but the plans for the lab are starting to come together.

Budget wise, I'm looking at about a hundred dollars a month in financial resources. I'll post a work up on the money side in detail once more details are available to me, as I'm not sure what qualities of equipment I'm going to need, or what I'll be able to improvise. With that said, I should have a fully functioning neurobiology lab in a year on the outside. More on this later.

Right now the bulk of my time is spent catching up my knowledge of research methods to my theoretical understanding. I've decided there are a few main avenues I need to pursue in the short term.

One is general knowledge of neuroscience. My sources of information have primarily been books aimed at giving a practicing clinician background knowledge on the fine workings of the brain. This is because up until about a year ago I was planning on getting my M.D. in psychiatry and going into research from there. Now, I've obviously become absorbed in pure research. Because of this my knowledge of brain chemistry is quite advanced, but my electrophysiology and macro scale anatomy is quite lacking.

The next is a need to be able to understand complex biology protocols. I've done some basic labs in the little bit of college I've had so far but it wasn't at nearly the level that I need to be operating at.

Along with this is the need to get a grasp on the general research methods of biology and specifically those which apply to neuroscience. My focuses for right now are reading everything I can about advanced microscopy and the optics and electronic/physical principles behind them, electrical sensor arrays and the electrical engineering behind them and cell culturing and manipulation techniques such as microfluidics.

An interesting non-neuroscience development in that I am going to be proposing a diybio genetics demonstration to a high school science teacher co-worker of my mom. I heard in passing that the teacher was planning on doing a tissue collection on her students and sending it in to be sequenced and analyzed by the Human Genome Project. While this is a great and innovative idea, I thought it would also be cool for the kids to be able to tangibly see and manipulate DNA as well. To any biohacker this immediately screams a coarse strawberry DNA extraction. I've put together the materials list, protocol, and some interesting background information that can be presented during the demo in the hopes that she will at least use the material and, ideally, allow me to conduct the demo under her supervision. My meeting is set for tomorrow, and you can bet that I'll post more when I know more.

That's about it for this missive from the lab in potentia, so I'll leave you with a few more links, this time basic background and technique articles.

Principles of Microscopy, an article by a professor of cell biology and molecular genetics explaining the principles behind visible light, fluorescence, confocal, and phase contrast microscopy.

Basic Microfluidic Concepts, an article on the University of Washington faculty pages site about the basic concepts behind microfluidics, as well as some numeric analysis of those principles.

Bonus: The beginners Strawberry DNA Extraction, from the University of Utah. Not really a protocol so much as a recipe (there's even meat tenderizer!), it's a great way to show kids and your friends the wonders of science.