The Interdisciplinary Laboratory

 Pre-Lab Assignment #2 for Experiment #8:
Genetic Map of a Bacterial Plasmid

NOTE: Please work with your lab partner to complete this pre-lab.  Each pair should submit one pre-lab.

At the end of the first laboratory period, you should have taken a photograph of your gel and labeled the lanes to indicate which lane corresponds to which digest or DNA size standard.  You will need this labeled photo in order to complete this pre-lab.

1. On your gel photo, locate the lanes that contain the two molecular size standards (the 100 bp and 500 bp "ladders").  The bands in these ladders represent DNA fragments of known sizes.  The 100 bp ladder contains DNA fragments in multiples of 100 bp from 100 bp to 1500 bp, with an additional band at 2072 bp.  The 600 bp and 1500 bp bands are doubly bright for easy identification (the 2072 bp band is also bright).  The 500 bp ladder consists of DNA fragments in multiples of 500 bp from 500 bp up to 8500 bp; the 2000 bp and 8500 bp fragments are doubly bright.  Compare the standards in your photocopy to the pictures in Appendix 8D of the lab manual.  The smallest fragments (100 bp or 500 bp) will have migrated furthest in the gel.  Identify each of the fragments in the two standards and write the fragment size (in bp) beside each band.

Now compare each of the bands in the lanes that contain digested plasmid DNA to the size standards.  Since linear fragments of equal size should migrate the same distance in the gel, you can roughly estimate the sizes of the fragments produced by the restriction digests by determining which fragment of known size migrated the same distance.

(a) For each digest, give the approximate size (in bp) of each of the fragments you obtained.
(b) Total the fragment sizes for each digest.  (If the digest was complete, the fragments should total to approximately 4361 bp, the known size of the plasmid.  Note that for these rough estimates an error of ± 500 bp in the total is acceptable.)
 

2. Next locate the lane containing your undigested plasmid control.  Remember that this will be a circular molecule and therefore it should have migrated further than a linear molecule of the same size.  Compare the band of circular DNA to the size standards.  The circular plasmid DNA migrated a distance comparable to a linear molecule of approximately what size?

[Note that the undigested control will also usually have a second, fainter band of large size (>4500 bp).  These bands are usually the result of two or more plasmid molecules interacting with one another to form plasmid dimers or other molecular complexes.]
 

3. Now reconsider any digests you listed above in (1b) for which the fragment size total was much greater than 4361 bp.  Do any of those digests contain a fragment that migrated the same distance as the undigested control?  If so, that fragment could be undigested, circular plasmid DNA.  If any digests contain fragments that are >5000 bp, they are probably plasmid dimers.  The presence of circular plasmid molecules in any of your digests indicates the digest is incomplete.

(a) Which (if any) of your digests appear to have undigested plasmid DNA?
(b) Subtract any bands that represent undigested plasmid or plasmid dimers from the totals you found in (1b).  What are the new fragment size totals for each digest?  Is the fragment size total for any digest still >4361?
 

4. During the next laboratory session you will have the opportunity to re-do any digests that appear to be very incomplete.  You should consider in advance which (if any) digests you might want/need to repeat, and plan accordingly.  In addition you will be able to re-run the results of the first week’s digests on gels of the same or higher agarose concentration to refine your measurements of fragment size.  A high concentration gel will yield better resolution and more accurate size estimates of small (<1000 bp) fragments.

(a) Which digests might you want to repeat?  Why?
(b) Which digests might you want to run on a high concentration agarose gel?  Why?
(c) Which might you want to run again on a low concentration gel?  Why?
 

5. Do you have any further questions about the background material, protocol or your results from the first week?