BMB lab exam 2 PSU

Which of the following will we be actually performing in lab this week? Select all that apply. Note: only select techniques that we will do, not techniques that were described in the experimental scenario as being completed prior to this lab.

 

Cloning the GFP gene into a plasmid using restriction enzymes and DNA ligase

 

Purifying plasmid from bacteria using a mini prep proceudure.

 

Doing a bacterial transformation reaction.

 

Using absorbance to analyze concentration and purity of a DNA sample.

B, C, D

An A260/A280 ratio of less than 1.8 implies what

likely contaminated with protein

What A260/A280 ratio implies pure DNA?

1.8-2.0

An A260/A280 ratio over 2.0 implies what

RNA contamination

recognition site for enzymes that can cut the DNA

xbal

element involved in controlling expression of genes in the plasmid

T7 promoter

antibiotic resistance gene

KanR

portion of plasmid that allows for the plasmid to be replicated when in bacteria

ori

sequence for a tag that gets added to protein and can help purify protein epxressed from this plasmid.

6X his

Rate the order of DNA in an agrose gel (which is fastest, slowest, middle)

Supercoiled is fastest, linear, nicked circles are slowest

What is the dye used to visalize DNA on agrose gel?

ethidium bromide

The ladder for agrose gel ranges from what to what

1-10 kb

Is the agrose gel used on DNA or Protein

DNA

Define a plasmid

small, double-stranded, circular pieces of DNA that is not part of the bacterial genome

Are plasmids part of the bacterial genome?

No

Plasmids encode for what three things?

Origin of replication, Antibiotic resistance, and Multiple cloning sites

To isolate the GFP gene from the bacteria what process do we use (what is it called)

mini prep

explain the four main steps of mini prep

Lyse the cells, spin down (pellet is bad, supernatant is good), spin down (DNA stays in the tube, liquid is thrown away), Elute the DNA

In mini prep cells are lysed via…

high pH

Xbal only leaves how many strands

1

Smal leaves how many strands

1

Xbal and Smal leave how many fragments

2

INCLUSION OF THIS GENE IN A PLASMID ALLOWS FOR SELECTIVE ISOLATION

antibiotic resistance gene (kanR)

RECOMBINANT DNA ALLOWS FOR…

gene cloning

THIS DNA CONFORMATION CONTAINS EXTRA TWISTS AND LOOPS THAT ALLOW IT TO MIGRATE FASTEST

supercoiled

BLUNT ENDS AND STICKY ENDS ARE ABLE TO BE ANNEALED WITH THIS ENZYME

DNA ligase

THIS STEP MUST BE DONE BEFORE PASSING THE LYSATE THROUGH THE SPIN COLUMN

centerfugation

FOR A MINI-PREP, YOU SHOULD AVOID THIS STEP AS IT CAN SHEAR GENOMIC DNA

vortexing

THIS FEATURE OF PLASMID DNA ALLOWS IT TO RENATURE AFTER EXPOSURE TO ALKALINE MOLECULES SUCH AS SDS AND NaOH

circular (only for plasmids, regular dna can’t do it)

THIS SOLUTION STABILIZES THE pH OF THE PROCESS AND PREVENTS NUCLEIC ACID DEGRADATION

TE buffer

THIS SPECIFIC ENZYME CLEAVES AT 4353 BP IN THE pET30 PLASMID

Smal

THIS SPECIFIC ENZYME CLEAVES AT 158 BP IN THE pET30 PLASMID

xbal

THIS SAMPLE IS USED TO SHOW THE PLASMID IN ITS UNCUT CONFORMATIONS

Control (uncut plasmid)

THE NUMBER OF FRAGMENTS PRODUCED DEPENDS ON THE NUMBER AND POSITION OF THESE

restriction sites

THIS PROPERTY OF THE GFP PROTEIN ALLOWS A VISUAL ESTIMATE OF PURITY/ISOLATION

fluorescence

THE GELS FOR THIS EXPERIMENT HAD THIS PERCENTAGE OF POLYACRYLAMIDE

12

THIS ELEMENT'S IONS HAVE A HIGH BINDING CAPACITY, ALLOWING PURIFICATION OF PROTEINS THAT BIND TO IT

Ni2+

THIS MOLECULE REMOVES THE REPRESSOR FROM THE LAC PROMOTER, ALOWING TRANSCRIPTION OF THE GFP GENE

IPTG (removed repressor)

THIS STRAIN OF E. coli ALLOWS FOR OVEREXPRESSION OF THE GFP PROTEIN

BL21(DE3)

ONLY THESE SAMPLES ARE LOADED ONTO THE Ni-NTA COLUMN

induced

Why are only induced samples loaded onto the Ni-NTA column?

The nickle grabs on to the his tagged protein

THIS MOLECULE COMPETES WITH HISTIDINE TO ELUTE GFP FROM THE RESIN MATRIX

imidazole

THIS FRACTION ISOLATES PROTEINS THAT DID NOT BIND TO THE RESIN

flow through

LAEMMLI SAMPLE BUFFER SMELLS BECAUSE OF THE PRESENCE OF THIS REDUCING AGENT

Beta Mercapotethanol

THIS PIECE OF EQUIPMENT VISUALIZES THE CHROMOPHORE PRESENT IN THE BARREL LOOP OF GFP

UV transiluminator

THIS SOLUTION CONTAINS A DYE THAT BINDS TO HYDROPHOBIC AND BASIC AMINO ACIDS IN PROTEINS

Sample buffer

THE DESTAIN CONTAINS THESE MOLECULES THAT REMOVE UNBOUND COOMASSIE DYE

acetic acid

SODIUM DODECYL SULFATE TAGRETS THIS PART OF THE PROTEINS IN SDS-PAGE

non polar/hydrophobic regions