Lab 10 Flashcards
what are some important rules to remember when handling a micropipette
- do not allow pipettes to come into contact with corrosive chemicals
- make sure the volume has been correctly set
- locking ring must be unlocked before changing volumes
how should hold the micropipette
keep it upright to prevent liquids from running inside the shaft
what kinds of tips are used with micropipettes
disposable ; tips can be autoclaved
what are some of the main structures on the pipettes
tip ejector button
digital volumeter
shaft tip holder
tip
what structure in DNA uses a covalent bond
the sugar-phosphate sides of the twisted DNA helix
what structures in DNA use a hydrogen bond to be held together
nitrogenous bases
why are the first three letters of the name of the enzyme italicized
refers to the bacterium from which the restriction enzyme was isolated
what does the fourth letter refer to
strain of bacterium if any
what do the numbers refer to
the sequence or order in which the enzymes were discovered
what forms the genetic code
the exposed bases
type II restriction enzymes
particular enzymes isolated from bacteria that can cleave the DNA of bacteriophages
how are cleavage sites determined
the enzymes recognizing palindromes of 5’ to 3’ and 3’ to 5’ sequences from four to several base pairs long
how can fragments produced by cutting double stranded DNA be separated
by size in a gel treated with an electric current
what is a palindromic sequence
nucleic acid sequence whereby reading in a certain direction (5’ to 3’) on one strand is identical to the sequence in the same direction (5’ to 3’) on the complementary strand.
what are the sticky ends of the type II enzymes
overhangs are called sticky ends because they can form base pairs with complementary DNA molecules
what does a restriction enzyme reaction contain
DNA to be analyzed
a restriction enzyme
restriction enzyme buffer mix
what is usually within a restriction enzyme buffer mix
- buffering agent to maintain constant pH
- salt to provide the correct ionic strength for the digest
- MG++ as a cofactor for enzyme activity
what is a “unit”
amount of enzyme needed to digest 1ug of the bacterial virus (lambda phage) DNA in 1 hour in a 50ul reaction
how many units do we usually use in lab
10-20 units
in the lab, what are treating the bacteriophage lambda DNA with
restriction enzymes EcoRI, Hindlll, and BamH1
what is gel electrophoresis
technique used to separate molecules on their sizes and/or charges
what is one of the materials the gel can be made out of
agarose
what is agarose gel electrophoresis
used to separate and analyze DNA and RNA
why does agarose gel electrophoresis sperate DNA molecules based only on their size
because DNA molecules are uniform in charge per mass
which way do DNA molecules migrate
towards the anode (positive terminal) because they contain negative charges
what is agarose
- polysaccharide
- purified from red seaweed
- forms a jelly like structure (gel) that contains holes & DNA molecules move through the holes under an electric field
what happens when you use more agarose
higher concentration, smaller the holes, and slower the DNA would migrate
how can DNA migration be sped up
with a higher electrical current
what are the two most important results from an agarose gel electrophoresis
size and amount of DNA
what does the position of the DNA indicate
size of molecule
- estimated by comparing to molecular marker loaded on same gel
what does the band intensity indicate
how much DNA within the band
- typically in ng or ug
what is a more qualitative way to analyze the sizes of DNA
make a plot
- semi log graph can be used to determine the sizes of DNA
what is the speed of migration of a DNA molecule based on
its size in kb/bp
How many positions would a plunger have
3
- rest stop
- 1st stop
- 2nd stop
why 37⁰C is chosen as the temperature for enzymatic reactions
It’s human body
temperature, usually the optimal temperature of an enzyme
