MOL BIO LEC MIDTERMS (The Blots)

1-14 Southern Blot, 15-18 Northern Blot, and 19-31 Western Blotting

Southern Blot

A molecular biology method to detect specific DNA sequences in a sample.

Restriction Enzyme (Endonuclease)

Cuts DNA at specific recognition sites. Incubation is at 37C. (Used in Southern Blot)

Gel Electrophoresis

Technique that separates DNA fragments by size using a gel-type substance.

Positive anode

DNA , RNA, and protein migrates in what anode.

Ethidium bromide

Stain used in Gel electrophoresis

Alkaline Solution (NaOH)

Used in the process of Denaturation in Southern Blot to separate DNA into single strands.

Neutralization

Process in Southern Blot that restores pH for efficient DNA transfer

Nitrocellulose nylon

In the process of capillary transfer what is used as the membrane where the DNA is transferred.

Wick

In Capillary Transfer this type of filter paper soaks the transfer buffer.

Capillary action

In the process of Capillary Transfer this moves the DNA to the membrane.

Radiolabeled (P-32) or fluorescent DNA

This is used as the DNA or RNA probe in Hybridization.

3x at 52C

In Hybridization how many times and at what temp do you wash the unbound probe.

42C Overnight

In Hybridization to bind the probe into DNA or RNA it must be done in what temp and time

Autoradiography

This process is for the detection of bands in the DNA or RNA.

Northern Blot

A molecular biology method to detect specific RNA sequences in a sample.

mRNA

In Northern Blot what type of RNA is analyzed to study gene expression

Formaldehyde

In RNA sample preparation this is used to prevent RNA secondary structures by maintaining its denatured state

Agarose-Formaldehyde gel

In northern blot what type of gel is used in Gel Electrophoresis

Western Blotting

Detects specific target proteins in a sample using antibodies.

SDS-PAGE

Separates proteins by molecular weight

Negative charges

In SDS Treatment, this denatures proteins and cots them with this to ensure all proteins have similar charge to mass ratio.

Coomassie Blue

in SDS-PAGE this is used for visualization of protein bands to see their sizes.

Marker Proteins

In SDS-PAGE this is used as size references.

Polyacrylamide (SDS-PAGE)

Gel type in Western Blot.

Nitrocellulose/PVDF

In Protein Transfer this is used as the membrane which binds the proteins tightly

Electroblotting

Technique used in Protein Transfer when using electric current to transfer proteins from gel to membrane.

Blocking

Process to prevent non-specific antibody binding in Western Blot.

5% Non-fat milk or BSA

Process of Blocking uses this type of agents

Primary antibody

This antibody binds specifically to target protein and incubated at 1-2 hours in room temp or 4C overnight

Secondary Antibody

Binds to primary antibody (anti-mouse IgG)

Horseradish Peroxidase

Enzyme producing chemiluminescent signal that is used in detection for Western Blotting