To Whom It May Concern:
I am a student doing a project on detmining the phylogenic relationships of different vegetables by comparing their genomes using gel electrophoresis. I have the procedure for the electrophoresis, but I am unsure of the procedure for extracting the DNA from the cells of the specimens. I also would like to know whether or not i need to use PCR to clone the DNA for the electrophoresis, or if I can get enough DNA just from the vegetables themselves.
Thank you for your aide. It is greatly appreciated.
Sincerely,
Kelsey Daly
Comparing the Genomes of Vegetables
Re: Comparing the Genomes of Vegetables
Kelsey,
you won't be able to diretly compare genomes by electrophoresis of extracted DNA- there are several ways to make comparisons by gel analysis: two options are 1) cut the genomic DNA with a series of restriction enzymyes and analyze the fragments by electrophoresis (agarose gels); 2) PCR specific regions of the genome and analyze amplified fragment size
one other thing to keep in mind is that agarose gels are usually visualized by ethidium bromide and UV light- both are dangerous. ethidium bromide is a carcinogen and needs to be disposed of correctly.
As for making DNA extracts, I assume you have access to the tools required. There are protocols are available on the web and from many lab web pages. A few places to start:
http://www.ucs.louisiana.edu/~mrv8332/H ... plants.doc
http://www.sidwell.edu/us/science/vlb5/ ... n_lab.html
http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb17/17053-1.pdf
You might also consider a bioinformatics approach- many plant sequences are available in public databases, and the degree of homology between either protein or DNA sequence is realtively easy to determine using freely-available tools.
you won't be able to diretly compare genomes by electrophoresis of extracted DNA- there are several ways to make comparisons by gel analysis: two options are 1) cut the genomic DNA with a series of restriction enzymyes and analyze the fragments by electrophoresis (agarose gels); 2) PCR specific regions of the genome and analyze amplified fragment size
one other thing to keep in mind is that agarose gels are usually visualized by ethidium bromide and UV light- both are dangerous. ethidium bromide is a carcinogen and needs to be disposed of correctly.
As for making DNA extracts, I assume you have access to the tools required. There are protocols are available on the web and from many lab web pages. A few places to start:
http://www.ucs.louisiana.edu/~mrv8332/H ... plants.doc
http://www.sidwell.edu/us/science/vlb5/ ... n_lab.html
http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb17/17053-1.pdf
You might also consider a bioinformatics approach- many plant sequences are available in public databases, and the degree of homology between either protein or DNA sequence is realtively easy to determine using freely-available tools.
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- Former Expert
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Experts,
I do indeed have the equipment to perform the DNA extraction. Thank yuo for this information. I intend to use restriction enzymes and measure the differences in the location of the DNA. Do you think that I will need to perform PCR in order to get enough DNA to perform the electrophoresis?
Thank you,
Kelsey Daly
I do indeed have the equipment to perform the DNA extraction. Thank yuo for this information. I intend to use restriction enzymes and measure the differences in the location of the DNA. Do you think that I will need to perform PCR in order to get enough DNA to perform the electrophoresis?
Thank you,
Kelsey Daly
Yep, you can extract enough to perform digests without amplifying, although you'll probably need to use a range of enzymes to get a good picture of species diversity- start with the 8 cutters and move down. Also, you'll probably need to use different extraction methods for high-yield recovery of different plants: high-starch veggies tend to require different protocols than the others.
best of luck,
htb
best of luck,
htb