cell and molec lab

Who invented PCR and when?

Kary Mullis in 1983

What is PCR?

A technique used to amplify specific DNA sequences.

Why is PCR used?

To make many copies of DNA for analysis

What are primers?

Short DNA sequences that define the region to be copied. Tells DNA polymerase where to start

What is the role of DNA polymerase?

Builds new DNA strands.

What type of polymerase is commonly used during PCR?

Taq

Taq polymerase

most heat resistant polymerase; building enzyme

PCR buffer components

Tris, KCl, and MgCl2

What is the role of the PCR buffer?

Maintains pH and optimal reaction conditions

What are the three steps of PCR?

Denaturation → Annealing → Extension

What happens during denaturation?

DNA is heated (~95°C) to separate strands; H bonds are broken

What happens during annealing?

Primers bind to complementary DNA sequences via the 3’ end

What temperature does annealing occur at?

~50–65°C.

What happens during extension?

DNA polymerase builds new DNA strands.

What temperature does extension occur at?

about 72C

What is mRNA?

Messenger RNA that carries genetic information from DNA to ribosomes.

What is RT-PCR?

A method that converts RNA into DNA (cDNA).

What enzyme is used in RT-PCR?

Reverse transcriptase

What is a key difference between DNA and cDNA

cDNA contains only exons (no introns).

Why does DNA move in a gel?

It is negatively charged.

Which direction does DNA move?

Toward the positive electrode (anode).

Which fragments travel farther?

Smaller fragments

What is TA cloning?

A method of inserting DNA into a vector using A-T overhangs.

What enzyme creates A overhangs?

Taq polymerase.

What is a plasmid?

A small circular DNA molecule in bacteria.

What is the origin of replication (ori)

Where DNA replication begins.

What is the multiple cloning site (MCS)

A region with restriction enzyme sites.

What is ligation?

Joining DNA fragments together.

What enzyme is used in ligation

T4 DNA ligase

What do blue colonies indicate?

No insert (lacZ active).

What do white colonies indicate?

Successful insertion (lacZ disrupted).

What is a restriction digest?

Cutting DNA with restriction enzymes

What enzyme was used for the restriction digest?

Ecor1

Why are HeLa cells immortal?

overactive telomerase

What protein is most abundant in the cytoskeleton?

Actin

What is phalloidin used for

Staining actin filaments.

What does DAPI stain

The nucleus

What is Western blotting?

A technique to detect specific proteins

What are the main steps of western blotting?

Separation → Transfer → Detection

What is SDS-PAGE used for?

Separating proteins by size.

What does SDS do?

Denatures proteins and gives them a negative charge.

What are the components of Laemmli buffer?

SDS, β-mercaptoethanol, glycerol, bromophenol blue

What does glycerol do?

Helps samples sink in wells

What does bromophenol blue do?

Tracks progress in the gel

What is the stacking gel?

Aligns proteins before separation

What is the resolving gel?

Separates proteins by size.

What does the primary antibody bind to?

target protein

What does the secondary antibody bind to?

primary antibody

What enzyme is attached to the secondary antibody?

HRP

What is β-actin used for?

A loading control; house keeping protein

If you want to run PCR on a DNA sample and you have a commercially available master mix that contains your DNA polymerase enzyme and appropriate PCR buffer. You have to add water, primer, and add your DNA template to the master mix as well. What is one more component that your master mix must contain besides the above for PCR to be successful

nucelotides

After 3 cycles of PCR, how many double stranded DNA molecules would you expect to be present

8

What can impact how big your DNA fragment will be in PCR

Where the selected primers will anneal

The amount of time selected for the extension cycle

DNA gel electrophoresis allows us to?

Detect and separate DNA molecules

What enzymes synthesis cDNA

reverse transcriptase

If you have a primer stock that is 30 uM. How much of your stock will you need to get a 1 uM concentration of the primer into a 50 uL PCR reaction volume?

1.67 uL

Within a gel electrophoresis, which way will the loaded gel run?

Towards the positive end

Is DNA positively or negatively charged?

Negatively

What determines how far a given sample will run down your gel?

Number of basepairs

Describe one difference between DNA and cDNA

cDNA only contains Exons

Describe one difference between PCR and RT-PCR

PCR takes DNA and makes more copies of DNA. RT-PCR uses RNA and makes cDNA

What are competent cells?

Cells that can readily take up exogenous DNA

Explains Antibiotic selection in detail

Only the bacterial cells that took up the plasmid containing the ampicillin antibiotic resistance gene will survive and grow, and those without it will not survive on the antibiotic-treated plate

Explains blue-white screening in details

• Begins with the lacZ gene
  lacZ encodes for an enzyme called β-
  galactosidase
  β-galactosidase cleaves X-gal, an analog of
  lactose
  When X-gal is cleaved, it yields a blue
  precipitate that can be seen by the naked eye

What color colonies would represent a successful transformation and why?

white because no β-galactosidase is produced, which means X-
Gal will not be cleaved, which means there will be no blue to be seen

What is transformation

The process of transferring exogenous genetic material to a bacterial cell

What enzyme mediates TA cloning

Taq Polymerase

Whats is restriction digest

The process of “cutting” DNA sequences

What are restriction enzymes? What and how did we use it in the lab?

Restriction enzymes cleave DNA into fragments at -articular sequences, we used ECOR1 which cleaves at both ends of our insert at 52 & 70 bp, adding 18 bp to GFP, making it 638 bp instead of 620 bp.

What is the flow of experiments to get to the final product

PCR→Gel Extraction & purification→Ligation→Transformation→Miniprep→Restriction digest→Gel electrophoresis for conformation.

How do phallodin interact with actin

They bind to f-actin subunits and prevents depolymerization of f-actin

What does DAPI allow you to visualize?

the nucleus

If you want to study the actin in the cytoskeleton what could you use

Phalloidin conjugated to a fluorophore

ORI

Where DNA replication begins
• ori sequences are generally high in As and Ts bc the two H bonds are easier to break

Antibiotic resistant region

Used for selection of plasmid-containing bacteria

MCS

• Segment of DNA that contains several restriction sites, allowing
for easy cutting by restriction enzymes and therefore easy
insertion of DNA
• Wherever the restriction enzyme cuts, our insert DNA will go in
between.

Before SDS-PAGE is run, our target protein samples are treated with _____, which makes the protein’s charge______.om sites

SDS;Negative

List the four ingredients that make up the laemmli buffer

SDS, BME, Glycerol, Bromophenol (Dye)

SDS

Denatures proteins, coats them in a negative charge

BME

- Denatures and breaks disulfide bonds

Glycerol

increases density so the sample loaded will sink to the bottom of the well

Bromophenol(dye)

tracks progress through the gel