IMED1002 Study Guide - Midterm Guide: Southern Blot, Thermal Cycler, Gc-Content
LECTURE 17 – MANIPULATING DNA I
Agarose gel electrophoresis is a technique that is used to separate DNA based on its size. A porous
gel (agarose) is placed in a tank and wells are made in it. DNA is added to the wells together with
tracking dye and glycerol which helps it become dense therefore sink to the bottom of the well. DNA
is negatively charged, therefore if we apply a current, it will migrate towards the positive anode.
Shorter DNA moves faster than longer DNA, therefore the smaller the length the DNA, the closer it
will be to the positive electrode. The migration speed also depends on the electric field strength,
buffer and % agarose in gel. A molecular size standard is also run on the gel – this is where we know
the size of the DNA fragments being run and this lets us work out the size of our unknown samples.
We can visualise the bands by using dyes that binds to nucleic acids and which fluoresce under UV
light.
Describe how dideoxy/Sanger sequencing works
PCR or the polymerase chain reaction is a technique used to amplify DNA. The sequence must be
known or between sequences that are known in order to design primers for replication. What you
need is your template which you want to amplify, two sets of primers - one for each strand, a heat
stable DNA polymerase (Taq Pol. is usually used), dNTPs for substrates, Mg2+, buffer to maintain pH
and an instrument called a thermocycler which heats and cools the reaction mixture.
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Document Summary
Agarose gel electrophoresis is a technique that is used to separate dna based on its size. A porous gel (agarose) is placed in a tank and wells are made in it. Dna is added to the wells together with tracking dye and glycerol which helps it become dense therefore sink to the bottom of the well. Dna is negatively charged, therefore if we apply a current, it will migrate towards the positive anode. Shorter dna moves faster than longer dna, therefore the smaller the length the dna, the closer it will be to the positive electrode. The migration speed also depends on the electric field strength, buffer and % agarose in gel. A molecular size standard is also run on the gel this is where we know the size of the dna fragments being run and this lets us work out the size of our unknown samples.
