Molecular Biology Lab 312L
Outline for Fifth Lab Period: Expt 12



Background:

    Mapping of the plasmid pBR322 has some similarities to the Lambda mapping homework of Experiment Three. We use a "ladder" of known fragment sizes to construct a standard curve, and determine the sizes of the pBR322 fragments produced by different restriction enzymes.

    However, in this experiment, we want more than just a size estimate for the pBR322 fragments. We are also trying to determine the orientation of the enzyme recognition sequences relative to each other. Then we will piece together the size and orientation information to construct a physical map of this plasmid.

    The experiment in the lab manual uses the Lambda HindIII digest to make the standard curve. That was fine for the mapping problem of Experiment Three, but not as useful for pBR322. Several of the pBR322 fragments are smaller than the last visible band on the Lambda HindIII digest (564 bp). For last year's Poster Project, the class tried to design an improved standard curve. We came up with two good candidates: Lambda DNA cut with PvuII, or Lambda DNA cut with StyI. This semester, we will test these two candidates and determine if they are an improvement over the Lambda HindIII digest.
 

Outline:

Work in groups of 3 or 4 (as directed by instructor). The group can work together through the entire experiment, including collaborating on data analysis.

Set up restriction digests of pBR322 (7 digests), Lambda HindIII digest, and EITHER Lambda PvuII OR Lambda StyI digest. The instructor will designate one of the two candidate enzymes for your group.
 

RESTRICTION DIGESTS:
Tube DNA 10x buffer EcoRI HincII PvuII HindIII StyI dH2O
E 3 ml pBR322 2 ml - H 1 ml         14 ml 
H 3 ml pBR322 2 ml - B   1 ml       14 ml
P 3 ml pBR322 2 ml - B     1 ml     14 ml
EH 3 ml pBR322 2 ml - MC 1 ml 1 ml       13 ml
EP 3 ml pBR322 2 ml - MC 1 ml   1 ml     13 ml
HP 3 ml pBR322 2 ml - B 1 ml 1 ml     13 ml
EHP 3 ml pBR322 2 ml - MC 1 ml 1 ml 1 ml     12 ml
l-HIII 6 ml l 2 ml - E       1 ml   11 ml
l-PvuII 6 ml l 2 ml - B     1 ml     11 ml
l-StyI 6 ml l 2 ml - F         1 ml 11 ml

  • Incubate tubes for a minimum of 30 minutes (60 is preferred) at 37'C.

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  • Cast a 1.0% agarose midigel.

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  • Add 4 ml of 6x Tracking Dye and electrophorese.

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  • Determine pBR322 fragment sizes based on the traditional Lambda HindIII ladder. Do a separate standard curve for the candidate enzyme (PvuII or StyI) and determine pBR322 fragment sizes. Make a table where you can compare the two values.

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