Restriction Digestion & Molecular Cloning
What's a recombinant plasmid?
The gene of interest is inserted into the plasmid by cutting at a specific site and the plasmid is known as a recombinant plasmid
What is needed for a PCR reaction?
DNA template
Taq DNA polymerase
dNTPs
Primers
What's agarose gel electrophoresis?
technique that allows us to visualize DNA sample and allows separation of a mixture of DNA fragments based on length and shape
What is DNA barcoding?
take a single gene to identify the species of an organism by comparing the DNA sequence to a reference database of such sequence
A DNA strand that is created using DNA pieces from 2 or more sources
What is recombinant DNA?
How can a scientist be able to analyze one gene among hundreds on a single piece of DNA?
By using restriction endonuclease, the scientist is able to cut the DNA at a particular point and observe only that.
When the temperature hits 45C in PCR, the reaction is in the ____ step where DNA strands ____.
Annealing
come back together
In nature, CRISPR (Clustered Regularly InterSpaced Palindromic Repeats), along with CAS9 proteins, refer to a system that functions as:
1. a viral infection mechanism
2. a plant cell immune system
3. a bacterial immune system
4. a bacterial cloning system
What is a bacterial immune system
True/False: There is no universal barcode for all life.
True; no true universal barcode for all life
What does RFLP stand for?
restriction fragment length polymorphism
What's the difference between sticky and blunt ends?
Blunt ends are non cohesive ends which contain paired DNA. Sticky ends have unpaired DNA which is overhanging.
True/False: DNA polymerase adds the free DNA nucleotides to the 3' end.
TRUE!
This will allow the new DNA strand to form. Nucleotides cannot be added to the phosphate (5') end because DNA polymerase can only add DNA nucleotides in a 5' to 3' direction. It has to be attached with sugar. Two strands of DNA are antiparallel.
Why does the gel need to be submerged in a buffer for the procedure?
ions will carry an electrical current through the gel
Another name for restriction enzyme
What is restriction endonuclease?
Fragments are sorted by this in gel electrophoresis.
What is size?
How is gene cloning done?
1.) Add bacterial plasmid, restriction endonuclease, and gene of interest into test tube
2.) DNA ligase binds insert and plasmid together
3.) Add buffer with cofactor (digestion) or add buffer with ATP (ligation)
4.) Cut insert with restriction endonuclease so it can be linear
5.) Do agarose gel electrophoresis to see how many bp and fragments
You can alter the % of agarose in the gel. So, by adding more agarose, you will make a firmer or softer gel?
FIRMER gel = smaller pores
tiny DNA = need high % of agarose
larger pores used for larger DNA = less agarose = flaccid gel = bigger pores
In biotechnology, the CRISPR-Cas9 system:
1. uses a 20 base-pair guide RNA
2. is used to edit a genome
3. all of the choices are correct
4. can be used to replace a damaged piece of DNA with a healthy piece
3. all of the choices are correct
The purpose of DNA ligase
What is recombining restriction fragments?
Restriction enzymes are called 'restriction enzymes' for this reason.
What is the enzymes restrict, or decrease, the effect of a virus on a bacterial cell?
What was the IV and DV of this experiment?
IV: recombinant DNA molecule
DV: # of bp present after digestion of recombinant DNA
What's the optimal temperature for Taq DNA polymerase?
72C
This is the optimal temperature at which Taq polymerase can add nucleotides to the hybridized primers to synthesize the new complementary strands.
How does cycle number affect the amount of DNA product?
# of new copies of DNA sequence of interest doubles after each cycle
Restriction fragments that have been cut on a zig-zag across a DNA strand?
What is a sticky end?
This is commonly used in genetic engineering.
what is bacteria?