Genetics Lab Midterm - Dr. White - The University of Findlay

BamH1 and EcoR1 jobs

cut the multicloning site of the plasmid; done to create an open DNA structure so other DNA can be inserted through a process called ligation

ligation

process in which DNA is inserted into an open part of a DNA structure

buffers

used to establish constant conditions in the reaction

mutation at codon 12

expression of a faulty protein where GGC (glycine) í GTC(valine) leading to amino acid glycine to be replaced by valine

point mutation

single nucleotide base substitution; alters protein structure and function

sticky ends

overhanging ssDNA on ends

annealing

process of creating dsDNA from complementary ssDNA; or the hydrogen bonding of 2 single DNA strands to form a double strand

Why do we heat ssDNA before cooling to anneal them?

to denature any hydrogen bonds formed from folding within itself to assure we can let the DNA anneal properly with a second strand of DNA

plasmid

circular piece of DNA

Poly-linker / multicloning site

part of plasmid DNA that has multiple restriction endonuclease sites that will allow for cutting and the inserting of foreign DNA into the plasmid

restriction endonuclease digestion

the cutting process of DNA

antibiotic resistance

a means to easily select bacteria that has taken up a DNA plasmid making it resistant to the antibiotic

blunt ends

DNA that is cut with no sticky ends produced

competent bacteria

bacteria that has been chemically treated to make them leaky and thus easier for DNA to enter through the outer membrane

How did we form competent bacteria?

treat bacteria with CaCl2, membrane has a - charge, so treatment takes calcium's +2 charge and makes membrane +, and now bacteria are competent

endonuclease

an enzyme that will cut DNA at a very specific sequence

transformation

term used to insert a plasmid into a bacterium; can bacteria take up plasmid? (little dots growing on the plate)

agarose

- more agarose = more fibers

-68 base pairs (small) so it slows down the DNA so it is not lost

Bromophenol Blue in 30% glycerol

loading buffer, dye

higher voltage in electrophoresis

faster the DNA will move, but the DNA will fray if it is moving too fast; slowing it down makes gel easier to read

Voltage higher than 90V in electrophoresis?

the DNA would fray and gel would be illegible

Why are enzymes added last?

to make sure all elements of reaction are catalyzed and so they do not denature

Buffer QG

mildly acidic solution, melts gel slice

Ethanol

precipitates DNA

Buffer PE

clean DNA by washing impurities through the membrane while keeping DNA stuck to the membrane

Buffer EB

remove DNA

heat shock of bacteria and DNA

disruption of membranes, allows plasmid to get inside bacteria

LB broth

growth medium for DNA

Log Phase

thin membrane, easily manipulated

Lag Phase

bigger membrane

Cell lysis solution (SDS)

detergent, breaks down phospholipids of cell membrane

GC repair

2 lines

AT repair

1 line

no repair

3 lines

Week 3 (purification)

getting rid of gel around DNA

100-degree water bath

get rid of secondary structures so it doesn't fold over on itself

37-degree (98.6 F - body temp) water bath

enzyme digest, bacteria growth

42-degree water bath

heat shock

70-degree water bath

annealing, bring down from 100 to 70, then let it cool from 70, bring it down slowly from 70 to let them anneal exactly the way we want them to

DNA color

pH, yellow is neutral

wash buffer

ethanol and alcohol, ethanol precipitates DNA

How did we know they annealed?

our double stranded annealed product is higher up the gel than the single stranded control

What does it mean to be anti-parallel?

parallel but moving in opposite directions

Why do we heat our DNA sequences when we add the G strand to C and the T to C and the T to G?

heating removes secondary structures in ssDNA that can occur due to Hydrogen bonding

Why do you keep your tubes on ice while waiting for the water to heat?

to keep things from denaturing

What would happen if the temperature drops too quickly after the hot water bath?

the DNA would fail to anneal properly

Why do we remove EcoR1 and BamH1 from pUC19?

in order to open the plasmid so we can insert our annealed sequences into the plasmid

What happens after the DNA of interest is inserted into the poly-linker region?

the plasmid is transformed into competent bacteria

Why is it that the only bacteria that takes up the plasmid DNA will survive?

because plasmids generally contain sites for antibiotic resistance

Why is it important that the bacteria is now resistant to ampicillin?

because all bacteria that don't contain the plasmid will be killed

How is it possible that bacteria is now resistant to ampicillin?

because the plasmid contains a genetic sequence which when transcribed and translated produces a protein that breaks down the antibiotic

When running a gel electrophoresis, why is it important to remember the phrase "run to red" in relation to where the DNA is added?

because DNA is negatively charged while the Red is positively charged, so we want the DNA to run to red so we can allow it to move through the gel

Why is it important to run only some of our annealed DNA?

to test the quality of our annealing reaction and to not jeopardize the annealed product

How does the mildly acidic solution melt the gel slice?

turns agarose into a soluble form

Why is the gel slice heated?

to aid in melting and dissolution of the gel

Why was it important to note the color of the melted gel?

to make sure it was at the correct pH

Why did we add isopropyl alcohol along with the buffer to the melted gel?

to precipitate the DNA and keep impurities in liquid form

How does the DNA bind to the membrane in the column?

buffer PE

Why is the speed important at what the collection tube is centrifuged at?

to assure that the wash solution goes over and through the DNA, and so the DNA is dried without coming through the membrane

What is the liquid in the collection tube? (Lab 5)

impurities and wash solution

Why was it important that the ligation was in the centrifuge overnight and not just as an in-lab centrifuge?

to keep ligation process slow for accuracy and time efficiency for the professor

Why do we heat shock the ligation+bacteria? And why for such a short amount of time?

disrupts the membranes to allow the plasmid to get inside the bacteria

What is added to the tubes to allow growth of the bacteria?

LB broth

What was the purpose of the glass beads and the ager plates? What role or purpose do they play?

beads distribute the bacteria across the agar plate; agar plate provides a growth medium

In what phase do we choose the bacteria in order to have competent bacteria? Why do we choose the bacteria here?

log phase because the membrane is thin and easily manipulated

How are plates placed in the incubator, and why?

- upside down to avoid condensation dripping on plate and interfering with the development on the plate

Lab 1

annealed DNA

Lab 2

cut plasmids and performed gel electrophoresis

Lab 3

inserted GT into the plasmid by ligation

Lab 4

insert plasmid into two different types of bacteria: mutant and wild type

Lab 5

two strains of bacteria were transformed with plasmid containing a GT mismatch to see which was proficient in DNA repair

Lab 6

recover plasmid from bacteria to measure the repair of defective DNA

How do we remove the plasmid from the bacteria?

centrifugation

Cell lyse solution was obviously used to break open the cell, how does it work or why does it work?

it is a detergent that breaks down the phospholipids of the cell membrane

How does TE buffer help to recover the plasmid?

elutes DNA from membrane

Why is there a significant difference between the G:T mismatch being repaired to G:C over the T:A?

if it is repaired to AT the NaeI site will not be present

Why use NaeI?

no recognition site on Puc19 for NaeI, that is specifically why we used it, designed specifically so if it repaired to GC, NaeI would then be a restriction site

Why does no repair give 3 bands?

there are three cuts the plasmid

How do you make a gel?

1. 4% agarose gel = 4g agarose per 100 mL 1X TAE Buffer

2. Use erlenmeyer flask and use 100 ml volume

3. Heat to boiling twice to get agarose into solution

4. Cool for 5-10 minutes

5. Cast gel in caster and insert comb. Allow to solidify.

6. If needed, fill 1x TAE buffer to fill reservoirs of apparatus

Incubator shaker

keeps at right temperature and agitation for bacteria to grow

Cooling centrifuge

keeps at 16 degrees to slow down ligation process

How are we sure that the plasmid doesn't self-ligate after we "open" it and before we insert our annealed products?

We cannot be 100% sure, however, accepted protocol suggests this is highly unlikely if the electrophoresis is moved on to fairly quickly as we do in lab

Why is Buffer EB noted to add directly on the membrane, and not on the side of the tube?

Its job is to release the DNA from the membrane and return it to the solution. It cannot do that if it is up on the walls of the tube.

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