MicroBio Lab Experiment 19 and 20 Plasmids and gel electrophoresis

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

1. 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

2. bind DNA

   transfer supernatant to the thermo spin column centrifuge 1 min

   discard flowthrough

3. wash the column

   add 500ul wash solution centrifuge then discard flowthrough

   spin again

4. 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