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plasmid vs chromosomal DNA
Plasmids are often used to study genes of interest and make mutations in genes using molecular techniques it is small circular, double stranded, extrachromosomal, and contains genes a cell might not always carry. to work with plasmid DNA must first isolate it from bacterial cells done by alkaline lysis miniprep protocol
chromosomal DNA is large, circular, double stranded, forms nucleosome, contains essential genes
plasmid mini prep outline steps
resuspend cells, lyse and neutralize them
add 250ul resuspension solution then vortex
add 250ul lysis solution invert 4-6 times
add 350ul neutralization solution invert 4-6 times centrifuge 5 min
bind DNA
transfer supernatant to the thermo spin column centrifuge 1 min
discard flowthrough
wash the column
add 500ul wash solution centrifuge then discard flowthrough
spin again
Elute purified DNA
transfer the column to new tube
add 50ul elution buffer and incubate 2 min
centrifuge 2 minutes then collect flowthrough
why do you add lysis after resuspending cells
this is to denature the protein cell walls to allow the plasmid DNA to separate from genomic DNA
do it after resuspension so that the lysis really gets the cells equal access
had to form a pellet so just the cells were in there
after adding neutralization a cloudy principate forms why
acidic buffer the plasmids zip back up but the genomic does not cause its a fatty
the buffer also has potassium which forces the genomic DNA to precipitate
why dont you want the white precipitate
its genomic DNA and proteins does not contain any Plasmid
plasmid is only in the supernatant
what does the wash step do
the plasmid still has residual molecules removes proteins and RNA
wash gets rid of it so only plasmid is left
Plasmid sticks to the silica in the little tube
what is the elution buffer for
to elute the plasmid DNA it is basic causes silica to become deprotonated and negative
the DNA is also negative so it elutes out
which way does DNA go in electrophoresis
the negatively charged DNA moves to the positively charges anode
agarose gel
is a natural polysaccharide made by dissolving the dry polymer in a boiling buffer than allowed to cool at room temp
when it solidifies it forms pores the DNA fragments are forced to move through these pores
what does the distance of the migration depend on
depends on the gel pore size and the DNA size
smaller fragments move through better
if you were to increase the concentration of agarose the pores will be smaller making fatty fragments move slower
what is high percentage agarose used for
the separation of small DNA molecules
high percentage can set very quickly and be uneven also brittle
most common percent is 1
what is low percentage agarose used for
large molecules
they are very soft and break easily
running buffer major roles
buffer ions are necessary to carry current flowing through gel
they prevent the damage of sample molecules by controlling the pH
buffer systems usually control the ionization state of sample molecules
the role of a loading buffer
these have dyes in them this helps to monitor the progress of the gel
the dyes are negatively charges so they move with the nucleic acids
the loading buffer also contains glycerol and sucrose which provide the nucleic acids with a density so the sample does not diffuse out of the gel
how do you estimate the size of the nucleic acid
by comparing the distance that it travels in the gel with the distance travelled by a marker or ladder
a marker or ladder is a DNA fragment of known size
preparing an agarose gel
agarose is dissolved in TBE buffer
heated to boil gelgreen stain is added then is let cool
held at 50 degrees
preparing TBE buffer
tris(base), boric acid, Na2EDTA, 1L water
pH is 8.3
assembling and pouring gel
casting tray flat rubber gaskets fit tight against wall
place comb in rig
let gel cool slightly and then pour into rig let cool and solidify
gel set up
pour some buffer on comb to loosen comb
buffer has lots of salt better wash quick so salt doesnt get stuck
pull casting tray out and rotate 90 degrees want wells on left negative side
fill the tank with TBE buffer gel should be fully covered
loading gel
micropipette into well
do not touch the gel the sample will sink so dont poke the bottom of well or sample wont be contained
imagining
drain buffer off casting train then plop the gel onto the uv image thingy then dispose
cleaning after electrophoresis
rinse everything with tap water
running buffer in waste
preparing plasmid DNA sample for loading
add water, loading buffer, plasmid dna
once finished pop spin it and throw her on ice
the concentration of pDNA in gel is approx 200ul
running the gel
should be run at approx 105V for 60 minutes
after 10 minutes make sure that the dye is advancing
bubbles should form at both ends of tank
gel green
stains the DNA and RNA
how do you determine the size of your plasmid
plot the distance migrated in mm on x axis and the size in bp on the y axis
then build curve using DNA ladder
