Molecular Biology Techniques Exam #1

How did we prepare the molecular standard, and why is it included?

Molecular standard mixed with loading dye containing glycerol so the sample sinks into the gel. It is included because it allows you to estimate the exact sizes of DNA fragments.

Define electrophoresis. Identify the components required to prepare a 1x TBE agarose gel.

Technique to separate charged DNA or proteins. You need agarose powder, TBE buffer, and acid stain.

Explain the roles of ethidium bromide, running buffer, and loading dye. Recall amount of loading dye used and why.

EtBr: a fluorescent stain and makes DNA fragments visible, an electrical conductor and chemical stabilizer, allows you to track electrophoresis. 2 pL of loading dye was added because its heavy density that increases the density of the DNA sample and best at tracking sample.

Which electrode does DNA migrate toward and why? What factors affect DNA mobility?

Towards the positively charged electrode because opposite charges attract. Size of shape of DNA.

Explain how restriction enzymes work and distinguish their general types. 

They are proteins that act as a defense against viral DNA through scanning and binding, cleavage, and cut ends. Type 2: HindIII, Type 1: large protein complexes, type 3: combo enzymes of different subunits. Type 4: target modified DNA.

Set up a 20 pl digest using 1x final buffer, enzyme, approx. 1 pg DNA< and water to volume.

1. Water- 23 pl. 2. Add buffer: 2 pl of 10x buffer. 3. Add DNA- 1 pg. 4. Add enzyme- 1 pl.

Review class example: 2 pl 10x buffer+ 2 pl EcoR1+ 1 pl DNA+ 15 pl water= 20 pl. Why does enzyme storage in glycerol matter?

It acts as an antifreeze and prevents ice crystals from forming and destroying the protein structure.

Identify reasons a digest may fail. Write an example of a six-base-pair palindromic DNA recognition site.

Missing buffer or contaminated DNA. 5’- G A A T T C- 3’, 3’- C T T A A G- 5’.

Predict restriction fragment sizes from linear and circular plasmid maps. explain your reasoning. 

1. Number of cuts (2- 1: up, 1: down). 2. Number of fragments resulting from a circular plasmid ( DNA cut n times, n fragments) . 3. vector backbone, gene insert (Mthfr)

Choose appropriate restriction enzyme to cut a target gene from a plasmid.

Extracting DNA from the gel.

Explain the purpose and major steps of gel extraction.

To isolate and purify a specific DNA or RNA after its been separated by size via gel electrophoresis.

Cut band, dissolve gel, bind DNA, add buffer, recover DNA.

Identify the desired end product of a bacterial plasmid mini-prep from lab 6

Purified, double stranded plasmid DNA (pAMP/ pKan).

explain the purpose of resuspension buffer, lysis buffer, and precipitation buffer

1. resuspends cell pellet and degrades RNA. 2. break cell membranes and denatures proteins/ chromosomal DNA. 3. Precipitation buffer: neutralizes high pH.

Explain why chromosomal DNA, proteins, RNA, and other membranes are not retained in the final plasmid preparation.

Chromosomal DNA, proteins, and cell membranes are denatured, dissolved and precipitated. RNA is digested by RNase.

define ligation and describe dna ligase's function. predict result of omitting ligase from a reaction.

1. The process of joining two DNA or RNA fragments together into a single continuous strand

2. An enzyme that cataylzes this joining process.

3. cannot maintain a stable plasmid and no colonies.

explain why both plasmids were cut with hindIII and bamh1 when transferring the kanamycin-resistance gene into pAMP. How did this generate the intended fragment?

Creating compatable sticky ends between them and makes sure the resistance gene can fit only in one direction of the backbone.

Both recognize specific sequences and flank the target genes on both plasmids.

Explain the role of magnesium chloride and calcium chloride in preparing chemically competent bacteria.

They provide positive ions of Mg2+ and Ca2+ and help shield negative charges of DNA and allow bacterial cells to take up foreign plasmid DNA.

define transformation. why are cells heat shocked and then allowed to recover for approximately one hour?

Transformation- bacterial cells take up foreign, extracellular genetic material from their environment.

At 37 C for one hour, it allows for the optimal growth of E.coli and ideal for repair cell membranes for transcription/translation.

Describe how ampicillin and kanamycin act, and how their corresponding resistance genes protect cells.

ampicillin targets the bacterial cell wall, while kanamycin targets bacterial protein synthesis.

Binds to the 30S ribosomal subunit that stops protein synthesis. The kanr gene produces an enzyme that chemically modifies by neutralizing kanamycin.

What are microsatellite colonies, why do they form, and how can successful plasmid uptake be identified?

Tiny bacterial colonies that form as a ring around a larger colony on a plate.

The main successful bacteria pump out a chemical that eats the medicine in the immediate area—> safe zone letting freeloader bacteria come and grow.

Look for large, primary colonies and confirm growth on AMP+ KAN plates.

explain the roles of X-gal and ampR.

X-gal: dye turns bacterial colonies blue (takes up plasmid), not: white

ampR: a gene that produces B-lactamase to destroy ampicillin, which allows bacteria to live on plate.