Really crappy biotech cue cards for last minute studying before midterm

Question 1

The resulting membrane, after capillary transfer contains what?

Answer 1

A replica of fragments originating from the agarose gel.

Question 2

What is the additional insurance used to immobilize DNA on the membrane?

Answer 2

UV crosslinking.

Question 3

Once the DNA is permanently bound, what is added to block it?

Answer 3

Salmon sperm DNA as the non-specific DNA source and skim milks as the inert protein.

Question 4

What is the point of blocking?

Answer 4

Inert proteins will occupy unbound sites to prevent background interactions.

Question 5

What are we using to block the membrane?

Answer 5

RapidHyb.

Question 6

What is the final step in the Southern blot?

Answer 6

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

Question 7

What is the purpose of adding formamide?

Answer 7

Used in the hybridization buffer to destabilize the secondary structures of ssDNA.

Question 8

What does the destabiliation afforded by formamide due?

Answer 8

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

Question 9

What is the purpose of polyethylene glycol?

Answer 9

Increase the speed of hybridization by reducing the concentration of available water in the hybridization solution.

Question 10

What does polyethelyne reducing water content effectively do?

Answer 10

increases the concentration of the biotin labeled prone and promotes faster hybridization.

Question 11

What determines DNA melting temperature and directly affects the binding of the ssDNA probe to complementary sequence immobilized on the membrane?

Answer 11

Temperature and ionic strength of the hybridization solution.

Question 12

The conditions of temperature and ionic strength in the hybridization solution are refferred to as?

Answer 12

the stringency of the reaction.

Question 13

What is high stringency?

Answer 13

high temperature and low ionic strength which results in a need for perfect complimentarity for binding.

Question 14

What is low stringency?

Answer 14

Low temperature and high salt concentration which results in not needed perfect DNA alignment.

Question 15

The detection of the biotin labeled probe is accomplished through?

Answer 15

visualization of the interaction with streptavidin via a chemiluminescent detection system.

Question 16

The streptavidin chemiluminescence system is very sensitive allowing for?

Answer 16

detection of picomoles of hybridized probe.

Question 17

Following hybridization, the blot is exposed to what?

Answer 17

streptavidin bound to HRP.

Question 18

Streptavidin binds to biotin causing permanent interaction with the ___DNA probe.

Answer 18

cDNA

Question 19

Visualization occurs because?

Answer 19

An enhanced luminol and oxidizing agent are added. Luminol interacts with HRP when oxidized and that results in light.

Question 20

Light solely comes from?

Answer 20

the probe binding to the target sequence.

Question 21

When the luminescent image is captured, what is interpreted?

Answer 21

MW and number of bands.

Question 22

The results will be used to identify if the digested DNA contained what?

Answer 22

The sequence of interest.

Question 23

What was the purpose of the PCR screen of genomic DNA?

Answer 23

To determined whether or not the plants gDNA contains a T-DNA insertion.