Gel electrophoresis?

Question

I need a project preferably at a high school level and above dealing with gel electrophoresis. It has to some how incorporate the use of restriction enzymes. Any help is definitely appreciated. 10 points to the best answer guaranteed. Thanks

Answer 1

Plasmid Mapping is good. I've have done it with high school students. It's fairly easy assuming you have have access to materials.

You will need:

Agarose gel electrophoresis gel tank and tray

Agarose

Power pack

Plasmids -puc19 is a good choice: http://www.fermentas.com/techinfo/nuclei...

Restriction enzymes

Water bath (or incubator)

Micro pipettors

Ethidium Bromide

UV light table

Digital camera and photo stand or a GelDoc

1. Digest plasmid DNA with various restriction enzymes. Set up several digests, some with single cuts, others with multiple cuts, and others with two enzymes. Use large amounts of plasmids for the digests and let them run a long time to ensure complete digestion.

2. Run the digestion products on on agarose gels to determine the overall size of the plasmid, and the relative locations of the RE cut sites.

Here is a more detailed description: http://www.carolina.com/category/teacher...

Note: Don't use Acrylamide Gels with inexperienced kids. Too much can go wrong (safety and set-up).

Note2: When picking your REs, don't choose ones that exibit star activity. It just confuses the results.

If you want any additional info, drop me an email. I've done this MANY times.

Answer 2

Hmmn, a project dealing with gel electrophoresis which includes the use of restriction enzymes eh?

Firstly let me say I'm not completely sure what kind of level I should be aiming this at, since you're being allowed to mess around with restriction enzymes and ethidium bromide I'm guessing you're probably old enough to work with bacteria as well.

What I'd suggest is that you take a plasmid containing a gene which confers resistance to an antibiotic (say kanamycin.)  You cut the gene from the plasmid using restriction enzymes, you can then isolate the gene by using electrophoresis and cutting the DNA spot from the gel (when you do the electrophoresis you should get two spots, a low weight spot which is the gene and a high weight spot which is the plasmid.)  Then you cut the gene from the gel and load it into another plasmid (making sure to cut the plasmid open with a suitable restriction enzyme) and then get some bacteria (say E. coli) to take up your plasmid.  If your experiment has worked the bacteria should be able to grow on agar containing kanamycin (if that's the antibiotic you went with.)

You'd have a number of points where you could do some elecrophoresis:

1. When you cut the antibiotic resistance gene from the first plasmid

2. When you cut the second plasmid

3. When you have inserted the kanamycin gene into the second plasmid

The idea behind this experiment is that you will be checking that the kanamycin gene got transferred using two different methods, gel electrophoresis and through bacterial culturing.

You can probably pick up everything you would need from a company called invitrogen.

If this is too simple / too advanced give me a shout and I'm sure I can help you design something suitable.