1. Plasmid Minipreparation of pAMP/pKAN Recombinants

Obtain your two tubes of overnight culture from the shaking waterbath. Label two eppendorf tubes "M1" and "M2". Keep your two samples separate throughout the procedure, and remember if you are isolating pAMP, pKAN, or pLIG (the potential recombinant).

Make the following changes in the protocol for Experiment 10a (minipreparation). The rest of the protocol will be the same as the lab manual:

A)  Use a P1000 to transfer all of the overnight culture to the eppy tube. Don't worry if there is less than 1 ml. Put the glass test tubes on a rack by the sink and remove the tape and the cap. Give them a squirt of 30% bleach and leave them on the rack to soak.

B)  For step #11, it is very important to avoid all the white cell debris or it will inhibit the restriction digest. Use a P200 twice to draw up the total 400 ul of supernatant, and wipe off the tip with a small piece of Kimwipe before transferring to a clean eppy tube.
Make sure the supernatant is completely free of particles. Spin and transfer a second time if you can see small pieces floating in the tube.

C)  Remember that step #12 is a carefully-timed step.

D)  At step #20, add 30 ul of TE to your DNA pellet instead of the 15 ul stated in the lab manual.
 

2. Restriction Analysis of Purified pAMP/pKAN Recombinants

Set up restriction digests:
Use a permanent marker to label four eppy tubes. Use the table below as a checklist when adding reagents.
 

Tube	M1	M2	5x Buffer E +RNAse	H2O	HindIII	BamHI
M1(-)	10 ul		4 ul	6 ul	0	0
M2(-)		10 ul	4 ul	6 ul	0	0
M1(+)	10 ul		4 ul	4 ul	1 ul	1 ul
M2(+)		10 ul	4 ul	4 ul	1 ul	1 ul

Add H2O, 5x buffer, and then DNA. Add enzymes last (keep them cold). Make sure digest reagents are mixed, and then incubate at 37'C for at least 30 minutes.

When it is time to load the gel, add 4 ul of 6x tracking dye to each tube. Load 20 ul of each sample. We will have separate gels for the pAMP, pKAN, and pLIG miniprep samples.

Your instructor will load the controls: undigested and digested pAMP, undigested and digested pKAN, and Lambda HindIII digest.