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When you run a gel of a plasmid that has been digested with a single enzyme for which there is a single restriction site on the plasmid why do you sometimes get more then one band?
Wiki User
∙ 14y ago
There are two possible reasons, and it may help enrich your digest concentrations by looking into these two possible solutions:
1. Incomplete digestion. The migration of an untreated plasmid travels faster that linearized DNA. The apparent band size of a circular DNA may be as much as 500bp smaller than its linearized counterpart.
2. Perhaps the agar wasn't fully melted when the gel was prepared. This can cause resolution problems, and sometimes a smear would show up.
3. The enzyme used is one of the more uncommon enzymes (noted by the volume the enzyme came in), and so these may be more picky at its turnover rate, leading to partial digestions.
Most of the time a restriction enzyme does its job really well if it was allowed to digest 1-2ug of DNA for at least 2 hours for NEB brand of enzymes. If it was just a diagnostic restriction digest, then you can predict what those bands would look like if it was a partial digest.
Good luck.
Wiki User
∙ 14y ago
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What biochemical tool would be use to cut a plasmid?
a Restriction Enzyme
Which enzyme would cut the plasmid without disrupting the function of?
Perhaps you mean a restriction enzyme, but not disrupting the function of whatever is not too clear. I think if you cut a plasmid with any restriction enzyme I am familiar with the function of that plasmid would be disrupted.
To produce a recombinant plasmid and the foreign DNA are cut with a different restriction enzyme?
Fasle.
Why is a restriction enzyme that cuts your plasmid more than once unusable?
Someone answer this already ;[
Which Restriction enzyme are studied in Recombinant DNA Technology?
It's not the restriction enzymes that are studied, its the DNA. The enzyme cuts or "restricts" the DNA strand at a known sequence of nucleotides. Different enzyme, different sequence. For a Biomanufacturing application, where we want to insert foreign DNA, the gene of interest is cut and spliced with a restriction enzyme into a recombinant plasmid, transformed into a bacteria, and sent merrily on it's way to make Insulin, or whatever. With an unknown piece of DNA (a functional gene that makes a protein of interest or is being studied), the plasmid has "restriction sites" or nucleotide sequences, for several restriction enzymes, all of which I have mapped out. The unknown piece of DNA is cut at each end by a single restriction enzyme and inserted into the plasmid, which gives me some landmarks. I insert the plasmid into a bacteria, grow a culture so the bacteria makes many millions of copies of the plasmid, extract the plasmid, and run an experiment called a restriction digest. The restriction digests are a series of reaction with single enzyme and combinations of two and three enzymes, all cutting the plasmid at different nucleotide sequences. Then I run an agarose gel electrophoresis, which separates all the different pieces of DNA by size, and do an analysis called a Restriction Map. This counts the DNA fragments and their sizes, which enzyme and combination of enzymes produced which sizes and how many fragments, which enzyme cuts where, which cuts were definitely in the known part of the plasmid, which were probably in the unknown DNA, adding up nucleotide sequence numbers to make sure different mapping guesses agree, etcetera, etcetera, and so forth. Until at last, a map of the size and restriction sites of the unknown DNA insert into the known plasmid vector is deduced. This used to be done by hand, but there are computer programs that do it now. This is Research, the Technology is down the line a few steps when the gene has been characterized, the protein produced has been characterized, the trials are done, and the restriction enzyme to insert the gene into the bacteria for Bioman has been established
What tool to use when cutting plasmid?
a Restriction Enzyme
What biochemical tool would be use to cut a plasmid?
a Restriction Enzyme
What tool will researcher use to cut plasmid?
They would use a Restriction Enzyme
Which enzyme would cut the plasmid without disrupting the function of?
Perhaps you mean a restriction enzyme, but not disrupting the function of whatever is not too clear. I think if you cut a plasmid with any restriction enzyme I am familiar with the function of that plasmid would be disrupted.
What is the biochemical tool that scientists use to cut plasmid?
They would use a Restriction Enzyme
To produce a recombinant plasmid and the foreign DNA are cut with a different restriction enzyme?
Fasle.
Why is a restriction enzyme that cuts your plasmid more than once unusable?
Someone answer this already ;[
Which Restriction enzyme are studied in Recombinant DNA Technology?
It's not the restriction enzymes that are studied, its the DNA. The enzyme cuts or "restricts" the DNA strand at a known sequence of nucleotides. Different enzyme, different sequence. For a Biomanufacturing application, where we want to insert foreign DNA, the gene of interest is cut and spliced with a restriction enzyme into a recombinant plasmid, transformed into a bacteria, and sent merrily on it's way to make Insulin, or whatever. With an unknown piece of DNA (a functional gene that makes a protein of interest or is being studied), the plasmid has "restriction sites" or nucleotide sequences, for several restriction enzymes, all of which I have mapped out. The unknown piece of DNA is cut at each end by a single restriction enzyme and inserted into the plasmid, which gives me some landmarks. I insert the plasmid into a bacteria, grow a culture so the bacteria makes many millions of copies of the plasmid, extract the plasmid, and run an experiment called a restriction digest. The restriction digests are a series of reaction with single enzyme and combinations of two and three enzymes, all cutting the plasmid at different nucleotide sequences. Then I run an agarose gel electrophoresis, which separates all the different pieces of DNA by size, and do an analysis called a Restriction Map. This counts the DNA fragments and their sizes, which enzyme and combination of enzymes produced which sizes and how many fragments, which enzyme cuts where, which cuts were definitely in the known part of the plasmid, which were probably in the unknown DNA, adding up nucleotide sequence numbers to make sure different mapping guesses agree, etcetera, etcetera, and so forth. Until at last, a map of the size and restriction sites of the unknown DNA insert into the known plasmid vector is deduced. This used to be done by hand, but there are computer programs that do it now. This is Research, the Technology is down the line a few steps when the gene has been characterized, the protein produced has been characterized, the trials are done, and the restriction enzyme to insert the gene into the bacteria for Bioman has been established
What must researchers know before they begin the process of gentic engineering?
you need to know which restriction enzyme to use. also, who is the doner and the plasmid.
What would happen if you cut both the jellyfish glo gene and puc18 plasmid with the ecor1 restriction enzyme?
If there is a EcoR1 site in either the middle of the Glo gene, or in the middle of the selectable marker site in the plasmid, it would likely disable either Glo, or the plasmid.
What is the function of restriction enzymes in the process of DNA recombination?
First, a specific enzyme is needed to cut the DNA from the donor genes at a specific site. This enzyme is called a restriction enzyme.The enzyme is used to cut out a piece of DNA that contains one or more desired genes from the donor's DNA. Next, a vector is needed to receive the donor DNA. Most frequently, a naturally occurring circular piece of bacterial DNA, called a plasmid, is used for this purpose. Finally, an enzyme is used to "stitch" the donor DNA into the plasmid vector. This enzyme is called ligase, and it creates permanent bonds between the donor DNA and the plasmid DNA. The result is that the donor DNA is incorporated into the bacterial plasmid, forming the recombinant DNA (rDNA)
An enzyme that cuts double-stranded DNA at specific nucleotide sequences?
Such an enzyme is called a restriction endonuclease
