Before starting the restriction enzyme digests first put your aliquots of lambda DNA, NEB buffer 2.1, StuI enzyme, and BsrGI enzyme into an ice bucket, and take this to your work area.
Label a tube as StuI – this will contain the StuI digest.
Label a tube as BsrGI to contain the BsrGI digest.
Then, label a tube S+B, to contain the digest with both of the enzymes.
First, pipette the 40 µL of water into each tube.
Then, using a fresh pipette tip each time, dispense 5 µL of buffer 2.1 into each tube and pipette up and down to mix.
Next, add 4 µL of lambda DNA to each tube, and then pipette 0.5 µL of each enzyme into the appropriate tubes.
Finally, add a sufficient amount of water to each tube to bring the total volume up to 50 µL.
Next, incubate the tubes at 37 ºC for 15 - 60 min in a water bath, and return the buffers and enzymes to the freezer.
To prepare an agarose gel to run the digest, in a 250 mL flask mix 1 g of agarose powder with 100 mL of TAE buffer.
Microwave the solution for approximately 90 s at full power, taking care to prevent the solution from boiling over.
Next, carefully remove the solution from the microwave using a glove or heat pad and place it to cool for about 15 min.
While the agarose solution cools set up a casting tray.
When the agarose solution is cool add 5 µL of 10,000X SYBR Safe gel stain to the solution.
Add the comb which is used to make the wells of the gel to the casting tray of the gel electrophoresis apparatus.
Pour the agarose gel solution into the casting tray and allow it to solidify.
Once the gel is solid, pull the comb straight up to remove it from the gel, and then remove the dams from the casting tray if appropriate.
Place the gel in the electrophoresis chamber with the wells positioned closest to the negative or black electrode and fill the chamber with 1x TAE buffer to completely cover the gel.
Remove your samples from the water bath.
Then add 8.3 µL of 6X loading dye to each sample.
Dispense 10 µL of the pBR322-BstN1 digest which contains DNA fragments of known size into the first well.
Then load 25 µL of sample StuI into the second well.
Place 25 µL of sample BsRGI into the third well.
Load 25 µL of sample S+B into the fourth well.
To run the gel plug the leads into the electrophoresis chamber and then into the power supply. The red positive lead should be at the end farthest away from the wells and the black negative lead should be nearest to the wells. This is because the samples will run from negative to positive.
Turn on the power supply and set the gel box to run at 170 volts for approximately 60 minutes.
After the run is complete turn off the power to the gel box and disconnect the electrodes from the tank.
Then remove the gel from the electrophoresis chamber and observe it with a UV transilluminator. CAUTION: Protect eyes from the UV by wearing goggles or using the provided shield.
Finally, use a cell phone camera to take a picture of the gel from directly above and parallel to the gel surface.