Which Of The Following Are The Approximate Sizes Of The Dna Fragments For The Mother?

Restriction Digestion/Gel Electrophoresis
Assignment i

With the combination of what yous have learned in the groundwork data and Experiments 1 and 2, you should be able to answer these questions. If you have trouble with something, refer back to the lessons and then effort over again. If y'all withal don't grasp the topic, either contact one of the people on the principal page, or try to find a real person to help yous (like an instructor or TA)!!

What we're going to do now is give you some experimental results and allow y'all interpret them, so let's jump right in. You have performed Restriction Digestion and Agarose Gel Electrophoresis on a plasmid you purified, using iii different Restriction Enzymes, and the gel is shown beneath. Unfortunately, you forgot to characterization your tubes or keep good records, and the only things you can remember about the experiment are that your standards are in Lane 5 and your uncut control is in Lane 1, and that you lot loaded roughly the same amount of total DNA in your sample lanes (1-4). Hey, at to the lowest degree you remembered that much!

1. Given no other data and using no math, approximately how large is your original plasmid?

35 kb
15.5 kb
vi.5 kb
5.0 kb
3.0 kb

Answer

2. How many times did the enzyme used in Lane 2 assimilate the plasmid? Does the data seem reasonable? What is the likely number of base pairs this enzyme recognizes?

Answer

iii. When Dna appears every bit a messy, continuous band every bit information technology does at the bottom of Lane 3, rather than contained, discreet bands, the effect is known equally smearing. What are some probable explanations for the smearing detected in Lane 3? Y'all should be able to come up with at least two.

Answer

iv. How many times did the enzyme used in Lane 4 digest the plasmid? Does the information seem reasonable?

Answer

Answers to Questions

1. Given no other data and using no math, approximately how big is your original plasmid? six.5 kb. Think that uncut Dna can exist plant in three forms, and the slowest of these is linear. Having only been cutting once, and therefore not having whatsoever DNA removed, the linear fragment in the uncut lane should gauge the size of the plasmid. Since it migrates near the half-dozen.56 kb standard lane (also a linear slice of Deoxyribonucleic acid), it is probable to exist virtually the same size.

Dorsum to Question 1

2. How many times did the enzyme used in Lane 2 digest the plasmid? Does the information seem reasonable? What is the likely number of base of operations pairs this enzyme recognizes? The enzyme digests the plasmid in two places. It is of import to call back most the state of the Deoxyribonucleic acid earlier digestion. The Dna used in this experiment was a plasmid, and plasmids are circular. If yous cutting a circle one time, you lot go 1 linear fragment. Yous must cut information technology a second time to get two linear fragments like in Lane 2. The data does seem reasonable because if y'all add up the guess sizes of the resulting fragments (roughly 4 kb and 2.5 kb), you get the original size of vi.5 kb. Lastly, it is likely that the enzyme used recognizes a sequence of 6 bases. six-cutters, if you'll recall, cut an average of one time every 4,096 bases. This is just an boilerplate, however, then in this instance where we take a piece of DNA 6,500 bp long, cutting twice is very reasonable.

Back to Question two

3. What are some probable explanations for the smearing detected in Lane 3? In general terms, smearing is when you take many bands together close enough in size that yous cannot distinguish betwixt adjacent bands (i.eastward., no resolution). With beginning molecular biologists, the nigh likely reason for the smearing is contamination by some stray nuclease that degraded the Deoxyribonucleic acid into dozens, hundreds, or fifty-fifty thousands of picayune pieces. Some other beginning mistake is to employ the incorrect buffer, incorrect temperature, or wrong conditions. Any or all of these could make the enzyme carry desperately, including cutting away at your DNA at multiple, random sites. However, equally you lot do more than and more than experiments like this, personal error becomes less of a concern and you need to start thinking in terms of the science. If this experiment was performed without significant error, the likely explanation is that a 4-base cutter was used. Cutting an average of one time every 256 bases in a 6.5 kb plasmid yields roughly 25 fragments, all smaller than the original. Information technology is unlikely that 1 could see 25 individual fragments of such a pocket-size size, and the smearing pattern is probably what would be detected.

Back to Question 3

4. How many times did the enzyme used in Lane 4 digest the plasmid? Does the data seem reasonable? If you said twice, you lot are correct, simply permit's encounter if you lot were correct for the right reasons. In question 2, it was pointed out that to get two fragments from a circular piece of Deoxyribonucleic acid, you demand two cuts. Then far so skilful. It was also mentioned that the total size of the resulting DNA fragments must add up to the original size. Uh oh--they don't, do they? Looking at the gel you see i band approximately 6.5 kb and one large ring at roughly 3 kb. Does 6.5 + 3 = 6.5? Not in this class.

Science doesn't lie, information technology's simply sometimes hard to interpret. And so interruption it down. Is there anything meaning most half-dozen.5 kb? Yes, information technology's the size of the original plasmid. This, plus the fact that in that location is a band in the uncut command (Lane one) which migrates to the same position, should suggest to you that not all of your Dna was digested (a common occurrence). This leaves the band effectually 3 kb. Could that band be 3.2 or 3.3 kb instead of iii.0? Hands. Unless nosotros plot a standard curve, we're merely approximating anyway. Is in that location annihilation meaning about 3.3 kb? Yes, information technology's about half of our original sample. If the enzyme cut the plasmid into two roughly equal sized pieces, those pieces would run about the same, and would likely exist indistinguishable on a gel. This is farther supported by the information most this experiment which states that roughly equal amounts of Deoxyribonucleic acid were loaded into Lanes one-four. Notice how much darker the 3 kb band in Lane 4 is than the bands in Lane ii. At that place is twice as much Deoxyribonucleic acid in that band than there is in either of the bands in Lane 2, and the data supports this conclusion.

Dorsum to Question iv

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