gels & rd Flashcards
why do we use gels
way to measure length of DNA
why do we need DNA length
determine if DNA is good enough to be sequenced
what length makes DNA sequencable
greater than 300 base pairs
properties of DNA
1) polar (soluble in water)
2) denatured & renatured
3) negatively charged
4) can be stained by Ethidium Bromide
5) reflects UV lights/rays
agar function in agarose gel
general medium for gels to travel through
buffer function in agarose gel
submerse gel and maintain charge distribution
loading dye function in agarose gel
glycerol makes DNA denser than buffer and agar, see the the sample in each well
ethidium bromide function in agarose gel
stain DNA
components of agarose gel
agar, buffer, loading dye, ethidium bromide
how to load gels
1) add loading dye to DNA sample
2) insert DNA into wells
3) insert a ladder
RUN THE GELS
will smaller or larger DNA fragments move further?
smaller fragments will move faster because there is more space within the agar to move
which end do you add DNA
negative end (cathode)
why do we use RD
a way to double check our PCR (insert size)
what is RD
uses restriction enzymes that cut DNA at specific points
what restriction enzyme do we use
PVUII (pvu2)
what are restriction enzymes
degrade foreign DNA in bacteria from viruses, cut at restriction sites
if RE degrades foreign DNA, why doesn’t it degrade bacterial DNA
bacterial DNA has methyl on nitrogenous bases
what site is our insert at
SfiiA and SfiiB sites
why do we not cut at the insert
less expensive, cut too close to insert might cut into insert
what do random cuts result in
smeared bands
how to do mockup
1) set gel picture to b&w (see bands better)
2) label ladder (left) and cut/uncut lanes
3) determine length of insert
what does the intensity/brightness of bands correspond to
size and number of fragments in DNA band
what will happen if you leave the enzyme in the plasmid for a long time
more plasmid it will cut
errors
1) partial digestion
2) contamination
3) lab error
4) puncture
how many base pairs are needed in PCR
300 base pairs
how many base pairs are needed in RDs
700 base pairs
where is Ethidium Bromide found
in gel (Kathie & Lina thought they had cancer when they touched the loading dye but it’s in the gel)
