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DNA Technology

How does a biologist propose marriage to his biologist girlfriend?

http://www.inquisitr.com/451957/biologist-proposes-tohis-girlfriend-with-dna-fragments/

Gel electrophoresis
Separates macromolecules based on rate of movement through an agarose gel in an electric field. Distance travelled 1/length

Animation! http://www.dnalc.org/resources/ animations/gelelectrophoresis.html

What size of DNA molecules can be separated? How are the electrodes of the electrophoresis chamber oriented relative to the starting point of the DNA samples? How are DNA fragments separated by size?

How are the separated fragments made visible?


What are DNA markers/ladders? In the resulting gel, what makes a band?

How do you get your DNA fragments in the first place?


www.biologyreference.com

Restriction enzymes/endonucleases
REs cut DNA molecules at a limited number of specific locations What are REs originally for? How is a bacterial cells own DNA protected from its REs? Terms: restriction site, restriction fragments, sticky end
www.goldiesroom.org

Animation! http://www.dnalc.org/resources/ animations/restriction.html

Restriction enzymes/endonucleases
Enzymes are named after the bacterium from which it is isolated

en.wikipedia.org

Which of the REs to the right produce sticky ends? Which produce blunt ends?
users.rcn.com

For this gel, those DNA fragments were obtained via PCR instead of using REs. PCwhat?
Theyre 5 sized PCR fragments (roughly 150, 300, 500, 700, 1kb), I went back through my notes to find 5 primer pairs that I knew worked pretty well (so dont feel bad, theyre selected out of primers that had been pre-validated =p). The other lanes are just mixes of the 5 sizes (either 2:3:4 or 4:6 volume mixes going in decreasing size, since larger fragments tend to be brighter). The gel actually didnt take that long (though it was terrifying loading it), but I made a mockup in Illustrator beforehand (along with a ladder to test what sizes to use), and then sketched it out beforehand so I knew what to add to each lane. - gdiguy2 (www.reddit.com/r/biology/comments/155fnj/there_is _literally_no_nerdier_way_to_propose/c7jnu8x)

Polymerase chain reaction (PCR)


Amplifying DNA in vitro
PCR can amplify any target sequence may times in vitro Lets summarize:
Step Denaturing Annealing Extension Temp (oC) Duration Whats happening?
Animation! http://www.dnalc.org/resources/ animations/pcr.html

What four ingredients do we need?


www.genomics.agilent.com

Gene Cloning in Plasmids


Amplifying DNA in vivo Producing many copies of a gene of interest was originally done in living cells. Terms: ampR gene, bacterial plasmid (cloning vector), gene of interest, hummingbird cell, hummingbird DNA fragments, lacZ gene, nonrecombinant plasmid, recombinant plasmids, restriction site, sticky ends
Why are bacterial plasmids widely used as cloning vectors?

Gene Cloning in Plasmids


Amplifying DNA in vivo Producing many copies of a gene of interest was originally done in living cells. Terms: bacteria carrying plasmids, colony carrying nonrecombinant plasmid, colony carrying recombinant plasmid, nonrecombinant plasmid, one of many bacterial clones, recombinant plasmids

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