Donna Almendrala

MCB 110L

September 12, 2007 - Experiment 7

Procedure I:

Goal – Digest the pET28-lacZ plasmid preps from lab 5 and then size the fragments on a gel.

1. For the pET28-lacZ, do 5 restriction digests using the following:

	Water (uL)	Buffer (uL)	DNA (uL)	Enzyme (uL)
Cla I		4: 2 + BSA	5	2
Sac I		1: 2 + BSA	5	0.5
Ssp I		2: 2	5	2
Hind III/EcoR V		2: 2 + BSA	5	0.5 + 0.5
Ssp I/EcoR V		2: 2 + BSA	5	2 + 0.5

2. Digest for about 1-2 hrs in 37˚C water bath.

3. Run the digests on a 1% agarose gel at 100V for an hour. Make sure to add a 1kb DNA standard lane. You can share a gel with another group.

4. Use 4uL of dye and 20uL of each digest for the lane.

5. After gel is run, photograph it and estimate the sizes of the DNA fragments.

6. Draw a restriction map for pET28- and deduce the orientation of the lacZ gene insert.

Expected Result – The gel should follow the same pattern as one of the mock gels shown on the previous page. Based on which mock gel it resembles, the orientation can be ascertained as either clockwise or counterclockwise.