Part I. Shown above are diagrams of four different plasmids carrying four differ
ID: 68852 • Letter: P
Question
Part I. Shown above are diagrams of four different plasmids carrying four different mammalian DNA sequences. For the first part of this exercise, two of the four plasmids above have been digested with two different restriction enzymes present in their multiple cloning sites. Following digestion, the resulting DNA fragments were separated by agarose gel electrophoresis in the presence of ethidium bromide and a photo of the gel was taken (see below). Your task is to determine which two of the four plasmids were digested. First, prepare a table of all available data for the four plasmids. Each plasmid should be a column in your table. Make separate rows for plasmid name, original plasmid size, DNA insert size, and total size of each recombinant plasmid. Then make additional rows for each restriction enzyme showing the size(s) of any fragments that would be expected following digestion with that enzyme. (Note: the total size of the MCS is so small that you don’t need to worry about it in your size estimates.) You will need the information from this table to analyze the gel photo and determine which plasmids were digested. The 1 kb DNA ladder in the gel at the far left and far right contains DNA molecules of known size (length). Those sizes are listed on the right of the gel. Use the DNA ladder information to estimate the size of each gel fragment in each of the plasmid lanes on the gel on the previous page (Note: the sizes of the uncut samples can not be estimated from the ladder, so just ignore those samples). Then make a table containing the data you obtained from the plasmid digest gel. Include plasmid #, estimated sizes of fragments in the gel for each enzyme used, and the identity of the two plasmids. Include a brief paragraph describing how you arrived at your decision.
1 kb DNA ladder uncut BamH EeoRI uncut BamHI Ecol!! I kb DNA ladderExplanation / Answer
NAME OF PLASMID
SIZE
SIZE OF INSERT
TOTAL PLASMID SIZE
pBS-NGFR
2960
950
3910
pBS-MBP
2960
1450
4410
P19-PLP
3950
1200
5150
pUC-SOX
2200
2200
4400
pBS-MBP and pUC-SOX are digested.
NAME OF PLASMID
SIZE
SIZE OF INSERT
TOTAL PLASMID SIZE
pBS-NGFR
2960
950
3910
pBS-MBP
2960
1450
4410
P19-PLP
3950
1200
5150
pUC-SOX
2200
2200
4400
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