Need help with hw problem DNA recombinant?

Question

need help with hw problem. I tried digested a recombinant pGEM with Sal1 restriction enzyme, and ran a gel to see the bands. I was supposed to see 2 bands but i only see one. I need to explain what two reasons why this occured can be, and how i can tell between the 2. Im thinking it can be a methylation problem, or just the enzyme not digesting properly??? Can anyone help please?? tnx!

Answer 1

1.  There is a possibility that you did not leave a sufficient amount of time for the digest to occur.  Therefore, you are seeing only a single band that represents the undigested plasmid.  However, this would likely result in a band that corresponds to a size less than the actual plasmid (since the plasmid will be supercoiled).

2.  (This is not a great suggestion, but it is a possibility).  You forgot to add the restriction enzyme into the digest.

3.  (This is the most likely possibility.)  The restriction enzyme has fully digested your plasmid into two bands, but the bands are very similar (or are the same) in size.  To resolve two bands that are similar in size (e.g. 800 and 900 b.p.), run the electrophoresis gel for a longer period of time and/or use a higher concentration of agarose in your gel.  If the bands sizes are identical (e.g. both 800 b.p.), it will not be possible to resolve the two bands.  However, you might choose to perform another digest using a different restriction enzyme.

I hope this helps!  If you are being marked on this h.w. problem, please write the answer in your own words.

Answer 2

recombinant DNA?.......a plasmid is taken from a bacteria and a part of DNA from another cell is inserted into the plasmid and then reinserted into another organism.... These scientist have made glowfish by doing that.........go to wikipedia and type in "recombinant DNA" and it will tell you all bout it...