Midterm 2 Flashcards
In the steps of cloning, what is Isolation done through?
PCR
True or False: Commomonas testosterone is viral
False
How large is the Genome of commonas?
5MB
Why is purification done after PCR?
to remove salt, enzyme (DNA polymerase), primers, dye, and dNTPS.
How do you measure the concentration of DNA?
OD
What concludes the Isolation step of gene cloning?
Purification and OD of PCR
How should a vector molecule be cut? Why not more than that?
Precisely at one position so new DNA can be inserted. A vector cut more than once will be broken into two or separate fragments and will be no use as a cloning vector.
Why does restriction work in preventing viral infections in bacteria?
Restriction occurs because the bacterium produces an enzyme that degrading DNA before it has time to replicate
What do Type 2 restriction endonucleases cut?
DNA at specific nucleotide sequences.
How do type II restriction endonucleases work?
each enzyme has a specific recognition sequence at which it cuts a DNA molecule.
What type of ends do restriction enzymes cut?
Sticky ends
What happens if sticky ends cut by restriction enzymes are complementary?
They can bind to one another
How do you calculate the number of recognition sequences for a particular restriction endonuclease?
4^(number of nucleotides it cuts at)
What is the pH most restriction endonucleases function at?
pH 7.4
What can happen to the restriction enzyme if the incorrect NaCl concentrations or Mg2 concentrations occur?
decrease the activity of the restriction endonuclease, they might also cause changes in the specificity of the enzyme
What is the conventional speed of a restriction enzyme?
1 unit of enzyme is defined as the quantity needed to cut 1 ng of DNA in 1 hour.
True or false: Temperature is important in consideration of restriction endonucleases.
True
How do you kill/inactivate a restriction enzyme?
a short incubation at 70°C OR addition of ethylenediamine tetraacetate (EDTA), which binds Mg2+ ions preventing restriction endonuclease action
Define electrophoresis
a technique that uses differences in electrical charge to separate the molecules in a mixture.
Why does electrophoresis work?
DNA molecules have negative charges, and so when placed in an electric field they migrate toward the positive pole .
What does the rate of migration of DNA in gel electrophoresis depend on?
shape
charge-to-mass ratio
Size
What size molecules of DNA travel the fastest?
smallest molecules
What are used as size markers in a gel electorphoresis? Is it precise
A DNA ladder; No, error of 5%
Is the size of fragment and rate of migration of a fragment to a gel proportional?
No it is logarithmic
What is the maximum length of fragment that can be used in gel electrophoresis?
30kb
What type of gel is used for small fragments? What type of gel is used for large fragments?
Polyacrylamide gel; Agarose gel
What is agarose and why is it used in gel electrophoresis?
Agarose is a complex polysaccharide extracted from algae. When solidified it creates a sieving matrix with pores the fragments can migrate to.
What is the relationship between concentration of agarose and size of pores?
Higher concentration smaller pores.
What is placed in the DNA sample prior to placing it in wells for gel electrophoresis?
bromophenol blue, and glycerol to increase density of solution so it stays in the wells.
What are the three conformations of plasmid DNA? Which one migrates the fastest?
Supercoiled, linear, and circular; Supercoiled
What type of electrophoresis is used for larger molecules? Name and explain it.
Pulsed field gel electrophoresis where the electrical field is repeatedly switched can be used for longer fragments of DNA. It may prevent trapping of DNA molecules.
What comes after purification and concentration of DNA in gene cloning?
construction of the recombinant DNA molecule.
How is a recombinant DNA molecule produced?
The vector and DNA to be cloned, are cut then joined together in a controlled manner.
What enzyme is used for cutting? For joining?
restriction endonucleases (for cutting) and ligases (for joining).
Name and define the four classes of DNA manipulative enzymes.
Nucleases: enzymes that cut, shorten, or degrade nucleic acid molecules.
Ligases: join nucleic acid molecules together.
Polymerases: make copies of molecules.
Modifying enzymes: remove or add chemical groups.
Name and define exonucleases and endonucleases
Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
Endonucleases are able to break internal phosphodiester bonds within a DNA molecule.
What in the function of DNA ligase in a cell?
to join together fragments of DNA arising from breaks in the cell such as okazaki fragments in DNA replication.
How does DNA polymerase I work?
usually prepared from E. coli. attaches to a short single-stranded region in a DNA molecule, and synthesizes a new strand, degrading the existing strand as it proceeds.
What are the two reasons DNA needs to be cleaved before being clones?
aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA,. gene will have to be cut out of the large DNA molecule
large DNA molecules may have to be broken down simply to produce fragments small enough to be carried by the vector.
Define restriction endonuclease
cleaves particular sequences (usually 4-8 base pairs)
How do cells prevent their own restriction endonuclease?
cleaving their own DNA so they are not recognized by the restriction endonuclease.
Why are type 2 restriction enzymes most commonly used in recombinant DNA research?
the have a high specificity for particular DNA sequences, and they cleave the DNA within or near the recognized sequence, and cut sticky ends
Name 4 properties of Type 2 restriction enzymes.
Require Mg for activity.
Cleavage sites with nearly all type 2 restriction endonucleases are palindromic DNA sequences.
Most recognize four or six base pair sequences.
Some create blunt ends and some sticky
What are DNA ligases used for. What energy source do they use?
They put together fragments with compatible 5’ or 3’ ends, by the formation of the deoxy pentose-phosphate bonds.
Use ATP.
Define ligation
joining two DNA fragments
What is done after isolation?
Ligation
What is needed for ligation
plasmid vector
ORF of the gene
T4 DNA ligase
Why is a plasmid used as a vector?
it can replicate without a host.
What does the T4 ligase do? Why is it called T4?
the enzyme helping joining the amplicon to the vector
Called T4 because it comes from bacteriophage T4
What temperature is the ligase reaction kept at
4C
Define vector
Carrier molecule
Where does the plasmid come from?
E.Xoli
Name three things the vector contains
Origin of replication
Ampicillin resistant genes
Restriction site
When do vectors turn into linear vectors?
when cut by a restriction enzyme.
How can the vector be reproduced?
Need to insert the vector into a host (bacteria)
What is amplification done through?
transformation
How large is the E.coli genome?
Genome is 5MB
True or False: The plasmid of E.coli is in the genome? How large is it.
False. 3kb-10kb
What is the plasmid? How many strands? Wat does it allow?
The plasmid allows them to have antibiotic resistance.
An extra chromosomal DNA.
Plasmid is double stranded
True or false: the plasmid in E.Coli has its own ORI.
True
How does the plasmid get cut.
has restriction sites so restriction enzymes can cut it.
What type of cell is E.Coli? What does this mean? How is it exploited?
Competent cell, easily pick up things. Exploit this to do transformation/amplification
What vector are we using for ligation? How is it supplied?
We use the PGEMT Easy Vector (3.1kb)
Vector is supplied as a linear vector, therefore requiring no cutting
What else besides all vector components doe the easy vector have?
LacZ’ and T overhangs
How does the DNA fragment bind to the vector?
Vector has T overhands and PCR produces produce with A overhangs. Ligase is the enzyme.
What is the molecule with the amplified DNA and the vector after ligation called?
recombinant DNA molecule.
What does ligation disrupt on the easy vector?
LacZ’ gene.
Where are the A overhangs on the PCR product and why?
A overhangs in the 3’ end because the 3’ end has hydroxyl which is easier to modify.
Why is the cloning we do called TA cloning?
A overhangs on the PCR product and the 3’ end and T overhangs on the vector join together using ligase.
Why do we need to do a transformation?
Due to needing DNA polymerase for replication of the vector, need to transfer to E.coli
What strain of E.coli are we using?
JM109 strain
What are the steps of transformation?
Place recombinant molecule and E.coli together so it is inserted into the E.coli.
Place both recombinant DNA (ligated product) and E.coli strain JM109 in a test tube.
True or false: In transformation the e.coli will pick up the DNA?
True
What happens to the E.coli after it ppicks up the DNA?
Ecoli will now have its own genome and the recombinant DNA.
How will the solution in transformation be kept?
The solution will be incubated at 37C with light shaking at 250rpm, and LB medium.
How often will every cell be doubled?
Ever 20 minutes, every cell will be doubled.
What happens when the host cell divides?
the genome and the recombinant DNA will double
What is needed for recombinant DNA replicate in its host cell? Why?
pressure. Need to grow the e.coli in ampicillin to motivate replication.
What occurs after incubation?
The solution is plated and kept overnight to grow colonies in the plate
What happens if the PCR primer is messed up?
DNA polymerase cannot work
The PCR does not work
What does polymerase di in the PCR
extends the primer complementary to the template strand.
Make the FP and RP for the following strands of DNA:
5’ - GACATATAGCTAGACTAC-3’
3’ - CTGTATATCGATCTGATG - 5’
FP: GAC ATA TAG CTA GAC 3’
RP: 5’GTA GTC TAG CTA TAT 3’
In DNA complementary nucelotide bonds are [] while adjacent bods are []
hydrogen; phosphodiester
How do you check the results of the PCR?
Gel electrophoresis using agarose
What percentage agarose was used in our experiment?
1%
What is the first lane in an agarose gel electrophoresis? What is is used for?
DNA ladder placed in the first well to help establish sizes.
What does the PCR product have that can show up in the bands? Ideally how many bands would you like? How many bands can happen?
genomic DNA, amplified primer, and primers; 1; 3
Why ideally would you want [] band?
Since it indicates primers attached and genomic DNA was amplified with primers attached.
Define DNA ladder? How much would you predict the band in the present experiment be? Does it give concentration?
Molecular weight marker to know the size of the fragment of the DNA.
774bp
No need to get OD.
What is used in OD?
Ultraviolet Light
Besides concentration, what can OD be used for measuring? How?
Protein contamination by measuring ultraviolight absorbtion of the protein.
What is beer’s and lamberts law
A=ecl
A is absorbance
e is absorbtion coefficient
c is molar concentration
l s optical path length
What value doe DNA absorb at? What about protein
260nm; 280nm
What shape is the graph? What does the peak indicate?
Bell curve; peak indicates level of absorption. Higher the peak at 260nm the greater the concentration.
