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).

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***

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.