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).
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