Biotechnology (W16) - Week 3-4 Background

Question 1

To perform analysis by southern blotting, DNA is initially what?

Answer 1

Digested by one or more restriction endonucleases.

Question 2

Restriction digest can be performed on which DNA types?

Answer 2

gDNA
PCR DNA
cDNA

Question 3

In this lab, we will be performing a restriction digest of what type of DNA?

Answer 3

gDNA

Question 4

Digested gDNA is then separated by what method?

Answer 4

Agarose gel electrophoresis

Question 5

The nucleic acids contained in the gel, post restriction digest, are ______ then transferred to what?

Answer 5

The nucleic acids contained within the gel are denatured and transferred to a positively charged nylon or nitrocellulose membrane.

Question 6

What transfer method will we be employing this lab?

Answer 6

Capillary transfer approach

Question 7

In terms of reliability, efficiency and speed, how would one describe transfer by capillary action?

Answer 7

Most reliable and efficient in terms of transfer.

Not the best in terms of speed.

Question 8

How does the capillary transfer approach work?

Answer 8

Blotting papers are used to wick transfer buffer through the gel, through the membrane, and into a stack of fibrous paper towel.
Eventually, either the wick becomes saturated with transfer buffer or the reservoir becomes exhausted.
As this occurs, the gel becomes dehydrated and the DNA is drawn out.
The nucleic acids are carried from the gel to the membrane where, movement is arrested due to attraction between the positively charged membrane and negatively charged DNA.

Question 9

The rate of transfer of the DNA fragments (using capillary transfer) depends on what?

Answer 9

The molecular weight of the fragment and concentration of agarose within the gel.

Question 10

Smaller DNA fragments are transferred from the 0.8% agarose gel to the membrane within ______ whilst larger pieces are transferred ________ (more than one word).

Answer 10

1. 1 hour

2. more slowly and less efficiently

Question 11

What is the concentration of the gel?

Answer 11

0.8% agarose

Question 12

How is the efficiency of transfer of larger DNA fragments accomplished?

Answer 12

Acid hydrolysis prior to denaturation. Essentially, larger pieces are hydrolyzed into smaller pieces, resulting in greater ease and speed of transfer.

Question 13

The resulting membrane contains a replica of the _________ originating from the _________ ___.

Answer 13

1. fragments

2. agarose gel

Question 14

The positively charged membrane allows for what with the nucleic acids?

Answer 14

Electrostatic interactions with the negatively charged nucleic acids.

Question 15

What must also be done to the transferred DNA to ensure that the ssDNA fragments are completely immobilized on the membrane?

Answer 15

UV-crosslinking

Question 16

Once the DNA is permanently bound, what is done to remove background interactions for chemiluminescence?

Answer 16

Pre-hybridization with a non-specific DNA and protein such that all unoccupied sites are covered/blocked from non-specific binding by probe molcules.

Question 17

To pre-hybridize, what commercially prepared product will we be using?

Answer 17

Rapid Hyb

Question 18

The final step of the Southern blot is what?

Answer 18

the hybridization of a labeled probe to the digested DNA at specific salt, pH, temperature and nucleic acid concentrations.

Question 19

What is the non-specific DNA in the rapid Hyb product? (milked em myself)

Answer 19

salmon sperm (Fkn nice m8)

Question 20

What is the non-specific protein in the rapid Hyb product?

Answer 20

Skim milk

Question 21

Typically, formamide is used in the hybridization buffer for what reason?

Answer 21

To destabilize the secondary structures of ss nucleic acids.

Question 22

Formamide effectively does what to the DNA and probe?

Answer 22

Lowers the effective melting temperature of the DNA and probe and permits lower hybridization temperatures from 65-68C.

Question 23

What is the purpose of polyethylene glycol?

Answer 23

Used to increase the speed of probe hybridization by reducing the concentration of the water in the hybridization solution.

Question 24

What does polyethylene glycol effectively do (by reducing the water content in the hybridization solution)?

Answer 24

increases the concentration of the biotin labeled probe and promotes faster hybridization

Question 25

What factors determine the DNA melting temperature and directly affect the binding of ssDNA probe to a complementary sequence immobilized on the membrane?

Answer 25

Temperature and ionic strength of the hybridization solution.

Question 26

What is the stringency of the reaction?

Answer 26

During nucleic acid hybridization or reassociation, the strictness to which base pairing is required under specific conditions of temperature, pH, salt concentration, etc.

Question 27

Conditions of high stringency would be what?

Answer 27

Requires all bases of one polynucleotide to be paired with complementary bases on the other.

Question 28

Conditions of low stringency allow what?

Answer 28

Some bases to be unpaired (between probe and DNA)

Question 29

What are permissive conditions of DNA hybridization?

Answer 29

low temperature and high salt concentrations.

Question 30

What are restrictive conditions of DNA hybridization

Answer 30

increased temperature and decreased salt concentration

Question 31

The detection of the biotin labeled probe is accomplished by what?

Answer 31

Chemiluminescence

Question 32

The chemiluminesence reaction involves which reagents and what are its benefits?

Answer 32

Involves the interaction between biotin and streptavidin-HRP and this method is extremely sensitive; allowing for detection of picomoles of hybridization probe.

Question 33

When is the blot exposed to streptavidin-HRP?

Answer 33

after hybridization

Question 34

Streptavidin binds to ______ causing permanent interaction with the ______ probe.

Answer 34

1. biotin (high affinity)

2. cDNA

Question 35

How does the luminol-HRP interaction work?

Answer 35

luminol interact with HRP and is then oxidized, resulting in the production of light.

Question 36

What is interpreted after the luminescent image is captured?

Answer 36

The molecular weight and number of bands hybridized to the probe

Question 37

Using the Southern Hybridization results, one will identify what?

Answer 37

If the digested DNA contained your sequence of interest.

Question 38

What will also be done to determine whether or not the plant(s) gDNA contains a T-DNA insertion?

Answer 38

perform a PCR screen of genomic plant DNA.