Lab 10 results Flashcards
restriction enzymes
- cut only some selective location
- found in nature (bacteria)
- they only cut if they see some sequence
palindromic sequence
5’ GAATTC 3’
3’ CTTAAG 5’
its the same cause you always read from the 5’ to 3’ end so they are still the same sequence
sticky ends
left side- G
CTTAA
right side- AATTC
G
single strands can stick to each other
blunt end
no single strand exactment; line cut right down the middle of the sequence
which type of end, sticky or blunt, is better for ligation
sticky end
how are restriction enzymes named
named after the bacteria they come from
- fourth letter represents strain
- first letter represents genus
what does the buffer contain
- some salt to control ionic strengths
what is the one of the most important salts in the buffer
Mg2+
make sures the enzyme is active
2 ul of 10X buffer meaning
10 times as concentrated as 1
unit of enzyme
- called a unit
- usually use 10-20 units
- don’t measure them by how much
what is an enzyme
protein so they can be denatured easily
why do we use 37 degrees C
- human body temp
- temp most enzymes are active in
what is in the loading dye/buffer
- chemical for controlling pH
- EDTA to stop DNA from breaking down by getting rid of Mg and Ca
- glycerol to make sample heavier so it will drop to bottom of well
why do you add the loading dye/buffer
- so you can see your sample drop in well
- dye migrates in same direction as DNA because both are (-)
- migrate at certain position depending on DNA
what is the purpose of gel electrophoresis
used to analyze DNA
what are some details of agarose
- not digestive
- when heated in water and then cooled, forms a gel and DNA can migrate through the holes
- polymer of carbs
what did we use to perform the agarose gel electrophoresis
buffer and agarose gel
if everything stays the same except agarose, what will occur
DNA will migrate to lower % agarose because DNA will move faster
if everything stays the same except for use of a different molecule, what will occur
small DNA fragment- migrate faster
large DNA fragment- migrate slower
if everything stays the same except for voltage, what will occur
higher voltage- DNA will migrate faster
too high voltage- melt gel
what does a low voltage usually provide
a slightly better resolution
what are some parameters affecting gel electrophoresis
- agarose %
- DNA fragment size
- voltage
why do we use SyBr safe
need something to stick to DNA to become visible
what percentage gel did we make in lab
1%
- usually universal concentration
- will analyze from a few 100 base pair to 100 kilo base pairs
0.7% gel
used for something bigger than 2 kilo base pair
2.0% gel
used for something smaller than 200 base pair
what happens if you make a gel higher than 2%
gel will become too sticky
what happens if you make a gel with too low of a %
gel can brake easily
what buffer can be used
TAE (EDTA) or TBE
TBE
used for small DNA
TAE
more universal
what end is DNA attracted to
the positive end since it is negative
- opposites attract
what does the restriction digestion buffer contain
Mg2+
when using a 5x stock buffer to make a solution of 20ul, how many ml of stock buffer is needed
4
if everything else remains the same, DNA migrates faster in agarose gel when
the voltage is higher
what is the least likely name of restriction enzyme
pfu
a typical agarose gel is what %
1%
what is the typical temperature for enzyme digestion in lab
37 degrees celcius
what does the loading buffer not contain
Mg2+
how is SyBr safe added
directly into the gel
