1. Bacterial Culture Techniques

Each group of three (or four) students:
 

Petri Plate:	MM294/pAMP	MM294/pKAN	MM294
LB + Amp (2 red lines)	Expt. (streak plate)*	Neg. control	Neg. control
LB + Kan (1 green line)	Neg. control	Expt. (streak plate)*	Neg. control
LB only (black or none)	Pos. control	Pos. control	Pos. control (streak plate)*

*Each member of the group should streak one plate for single colonies, using either the pattern suggested in class, or in the lab book (pg. 18).

Label the petri plates properly:

Label the bottom of a petri plate (the half with the agar). Label along the outside edge so you can see the colonies in the middle. Put your lab day, initials of your group, and the strain of bacteria that you put on the plate (MM294, MM294/pAMP, or MM294/pKAN)

2. Measurements, Micropipetting, and Sterile Technique

Each student will practice with the small and large volume Micropipettors. Try both the Pipettman and V3 models (share them).

Do the exercises outlined in Lab 1, Part II and Part III. You should do at least one exercise with each pipettor size.  Check your volumes to see if they add up to the correct total. Take as much time as you need to learn; this is important for everything we do.

Do these two exercises using the P200:

Tube	Sol. I	Sol. II	Sol. III	Sol. IV
G	60 ul	30 ul	40 ul	70 ul
H	50 ul	40 ul	80 ul	30 ul

When you are confident that you can accurately measure 10 uL, come demonstrate to me. When I give you permission, you can set up the restriction digests outlined below.
 

3. Restriction digestion of pAMP and pKAN plasmids

Each student will set up the following restriction digests:
 

Tube:	DNA	10x buffer E	10 U/ul mix of HindIII + BamHI	dH2O
pAMP digest	4 ul pAMP	2 ul	1 ul*	13 ul
pKAN digest	8 ul pKAN	2 ul	1 ul*	9 ul

*NOTE : Your Instructor will add the restriction enzymes to the digest.
 

Follow these instructions to set up a restriction digest:
 

• Add the dH₂O first; can use the same tip.

• Add the buffer; can use the same tip because buffer is the same.

• Add the DNA; use a NEW tip each time (note that we have two different types of DNA).

• Tubes must be clearly labeled with pAMP or pKAN and also with your initials.

• Add enzyme last (bring the tubes to your Instructor).

• Incubate at 37' C for 40-60 min.