Spin falcon tubes @7600rpm, @4C, for 5min to pellet
Discard supernatant by decanting
Reconstitute pellet in 250μl cold Buffer P1 and transfer to microcentrifuge tube
Add 350μl Buffer P2, invert 4-6 times to mix thoroughly
Let stand for less than 5 minutes
The timing for the next few steps is important. Don’t delay.
Add 350μl Buffer N3, immediately invert 4-6 time to mix thoroughly
This step will form a white precipitate
Immediately spin @max speed for 10min @room temperature
Pipette supernatant into spin column while avoiding the precipitate
Centrifuge 60sec, discard flow-through
Add 750μl Buffer PE to column, let sit for 60sec, spin 60sec
Discard supernatant and spin another 60s to dry
Transfer to clean microfuge tube and let sit 60s
Add 30 or 50μl qH2O and let sit 60s, spin 60s
30 results in higher concentration but lower total yield
Pipette and reapply flow through, sit 60s, spin 60s
Nanodrop Every time you finish a reaction (and cleanup) you should check to see how much DNA you actually have in your test tube before preceding to the next step. This ensures that reactions that require specific molar ratios (ligations) are run correctly .
Procedure
Make sure you have a pipette that can measure 1μL and an aliquot of your elution buffer
Lift the arm on the nanodrop and load 1 μL of your water/elution buffer aliquot onto the small, silver well (pedestal). Gently close the arm, then reopen the arm and dab the blank liquid off the machine with a kimwipe. Make sure you wipe off the metal nubbin on the arm of the machine, too. Put the arm back down. All this ensures that the nanodrop reading area is clean to begin with
Turn on the computer and open “Nanodrop 2000”
Select “nucleic acid” from the opening menu
A window will pop asking if you want to add this data to the previously saved file. Don’t unless you were the previous user.
The nanodrop will perform a self calibration test for a few seconds
Select the appropriate type of nucleic acid you have in your sample from the dropdown menu. Most likely this is DNA.
Next you need to run a blank. Again load 1μL of your elution buffer onto the pedestal, but this time while the arm is down click the "blank" button on the screen. Wipe down the machine.
Load 1 μL of your sample. Gently close the arm and click the “Read” button
Let it read. If you clicked to create a new file in step 3a, then it will ask you for your filename info and stuff like that. Go through that.
Finally, the results should pop up on the graph and the table below it. Make sure the graph has a good 260/280 ratio (usually greater than 1.75). The graph should have a pronounced peak in the left-center of the plot, and should be pretty low on the right side. The curve of the graph should look relatively smooth.
Generally, the quality of the read should be very high for something like a miniprep and will often be much lower when reading the product of a PCR or digest cleanup
If the graph quality looks pretty good/normal, take note of the "ng/μl" value returned; this is the relevant information giving you the concentration of DNA in your sample
Repeat the scan (literally just click the "Read" button again) 2-3 more times to ensure that the read is consistent, and average the value
Save your data somehow (I just write it down, but you can screen capture if you want) and make sure to write the value on the tube containing the sample, wipe down the nanodrop, gently lower the arm, quit the program, and shut the computer. School’s out, you’re done!
Digestion
20 μL Recipe for any combination of the EcoRI, XbaI, SpeI, PstI:
500-1000 ng DNA (as close to 1 μg as possible)
0.5μL Enzyme 1
0.5μL Enzyme 2
2μl appropriate buffer (see NEB doubledigest finder, but for most biobrick enzymes, CutSmart will work perfectly)
Top up with qH20
Mix reagents, adding enzymes last
Incubate at 37°C for 1-2 hrs (<30 min for HF)
Heat kill at 80°C for 20 minutes if proceeding to ligation
Verification
Gel Casting: 0.75% agarose (if DNA>1000bp)
40mL 1x TAE
0.3 g agarose
1 aliquot (~5μl) gel red
Add dry agarose to clean bottle (small enough to fit in microwave)
Add 40mL 1x TAE buffer
Microwave with cap on but loose, swish periodically, until solution is clear and smooth
Pipette in gel red, directly into solution (heat stable so don’t worry about the temperature)
Pour into gel tray, making sure that tray is oriented and tightly inserted such that leaks will not occur, and that the gel is level
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