Lab 4: SDS Page Analysis

Question 1

What is the purpose of SDS PAGE analysis?

Answer 1

To confirm protein expression and purification by visualizing expressed proteins and estimating their molecular weight.

Question 2

What is the composition of the 12% resolving gel for SDS PAGE?

Answer 2

• 30% acrylamide mix (40:1)
• 1.5M Tris.Cl pH 8.8
• 10% SDS
• 10% ammonium persulphate
• TEMED

Question 3

What is the pH of the Tris buffer used in the stacking gel?

Answer 3

pH 6.8

Question 4

Fill in the blank: The stacking gel is prepared with _____ acrylamide mix.

Answer 4

30%

Question 5

What is the volume of the stacking gel prepared in the protocol?

Answer 5

3ml

Question 6

What is the purpose of overlaying the gel mix with 0.1% SDS solution?

Answer 6

To allow the gel to set properly.

Question 7

What should be done after the gel has set for at least 20 minutes?

Answer 7

Pour off the 0.1% SDS overlay and mix and add the stacking gel.

Question 8

What type of buffer is used to fill the inner and outer buffer chambers?

Answer 8

Tris-glycine buffer

Question 9

At what voltage should the samples be electrophoresed?

Answer 9

100V

Question 10

What is the purpose of the bromophenol blue dye in the electrophoresis process?

Answer 10

To indicate when the samples have reached the bottom of the gel.

Question 11

What is the staining solution used after electrophoresis?

Answer 11

1% commassie blue stain

Question 12

How long should the gel be stained with commassie blue stain?

Answer 12

1 hour

Question 13

What is done to destain the gel?

Answer 13

Transfer to a solution lacking commassie stain for several hours, changing the buffer after one hour.

Question 14

What type of disorder is Prader-Willi syndrome?

Answer 14

Imprinting disorder

Caused by failure to express the SNRPN gene product.

Question 15

Which gene is associated with Prader-Willi syndrome?

Answer 15

SNRPN (small nuclear ribonucleoprotein-associated polypeptide N)

Expressed only on the paternal chromosome 15q11.

Question 16

How is the maternal SNRPN gene affected in Prader-Willi syndrome?

Answer 16

Methylated and not transcribed

This leads to loss of expression of the SNRPN gene.

Question 17

What genetic events can lead to Prader-Willi syndrome?

Answer 17

• Deletion of paternal allele
• Maternal isodysomy

Both lead to loss of expression of the SNRPN gene.

Question 18

What type of disorder is Angelman syndrome?

Answer 18

Imprinting disorder

Caused by failure to express the UBE3A gene product.

Question 19

Which gene is associated with Angelman syndrome?

Answer 19

UBE3A

Expressed only on the maternal chromosome 15q11.

Question 20

How is the paternal UBE3A gene affected in Angelman syndrome?

Answer 20

Methylated and not transcribed

This leads to loss of expression of the UBE3A gene.

Question 21

What genetic events can lead to Angelman syndrome?

Answer 21

• Deletion of maternal allele
• Paternal isodysomy

Both lead to loss of expression of the UBE3A gene.

Question 22

What technique is used for diagnosis in the provided content?

Answer 22

Southern blotting and methylation sensitive restriction enzymes

This technique is utilized to analyze DNA fragments for genetic disorders.

Question 23

What is the characteristic of Not 1 enzyme?

Answer 23

Methylation sensitive

Not 1 recognizes and cuts methylated DNA.

Question 24

What is the characteristic of Xba 1 enzyme?

Answer 24

Methylation insensitive

Xba 1 does not recognize or cut methylated DNA.

Question 25

What is the size of the DNA fragment obtained from cutting the paternal chromosome 15q11 with Not 1/Xba 1?

Answer 25

0.9kb ## Footnote This fragment size is indicative of Prader-Willi Syndrome.

Question 26

What is the size of the DNA fragment obtained from cutting the maternal chromosome 15q11 with Not 1/Xba 1?

Answer 26

4.3kb ## Footnote This fragment size is associated with Angelman Syndrome.

Question 27

In the context of Prader-Willi and Angelman syndromes, what does a 0.9Kb fragment indicate?

Answer 27

Prader-Willi Syndrome (PWS) ## Footnote The presence of the 0.9Kb fragment suggests a paternal deletion.

Question 28

In the context of Prader-Willi and Angelman syndromes, what does a 4.3Kb fragment indicate?

Answer 28

Angelman Syndrome (AS) ## Footnote The presence of the 4.3Kb fragment suggests a maternal deletion.

Question 29

True or False: Cutting the paternal chromosome 15q11 yields a larger fragment than the maternal chromosome when using Not 1/Xba 1.

Answer 29

False ## Footnote The paternal chromosome yields a smaller fragment (0.9Kb) compared to the maternal (4.3Kb).

Question 30

Fill in the blank: Cutting maternal chromosome 15q11 with Not 1/Xba 1 yields a _______ fragment.

Answer 30

4.3kb ## Footnote This fragment size is significant in diagnosing genetic syndromes.

Question 31

What is Southern Blotting?

Answer 31

A method used to detect specific DNA sequences in DNA samples ## Footnote It involves transferring DNA fragments from an agarose gel to a nitrocellulose membrane.

Question 32

What is the purpose of size markers in Southern Blotting?

Answer 32

To determine the size of DNA fragments ## Footnote Size markers are DNA fragments of known lengths used for comparison.

Question 33

What type of gel is used in Southern Blotting?

Answer 33

Agarose gel ## Footnote Agarose gel electrophoresis separates DNA fragments based on size.

Question 34

What enzyme is typically used to digest DNA in Southern Blotting?

Answer 34

EcoRI ## Footnote EcoRI is a restriction enzyme that cuts DNA at specific sequences.

Question 35

What is the process of transferring DNA fragments to nitrocellulose paper called?

Answer 35

Blotting ## Footnote This step involves the transfer of separated DNA fragments from a gel to a membrane.

Question 36

What solution is used to separate DNA fragments during the blotting process?

Answer 36

Alkali solution ## Footnote An alkali solution is used to denature the DNA and facilitate transfer.

Question 37

What is the role of a labeled DNA probe in Southern Blotting?

Answer 37

To hybridize with complementary DNA bands ## Footnote Labeled DNA probes are used to detect specific sequences on the membrane.

Question 38

What technique is used to visualize the labeled DNA probe?

Answer 38

Autoradiography ## Footnote Autoradiography allows for the detection of radioactive or fluorescent labels in the DNA.

Question 39

Fill in the blank: The DNA fragments are separated by _______ electrophoresis.

Answer 39

agarose gel

Question 40

True or False: Southern Blotting can only be used for labeled DNA.

Answer 40

False ## Footnote Southern Blotting can be performed with unlabeled DNA, but labeling enhances detection.

Question 41

What is the term for the process that modifies mature RNA?

Answer 41

Splicing ## Footnote Splicing is a crucial step in RNA processing where introns are removed, and exons are joined together.

Question 42

What does SNRPN RT PCR indicate when absent?

Answer 42

Diagnostic ## Footnote The absence of SNRPN in RT PCR can be indicative of certain genetic conditions.

Question 43

What percentage is referenced in the context of SNRPN RT PCR?

Answer 43

82% ## Footnote This percentage may refer to the sensitivity or specificity of the SNRPN RT PCR test.

Question 44

Fill in the blank: _______ is always present in the WASP control.

Answer 44

WASP Control

Question 45

What type of nucleic acid is associated with SNRPN?

Answer 45

RNA ## Footnote SNRPN (Small Nuclear Ribonucleoprotein N) is involved in RNA processing.

Question 46

What does the acronym RT PCR stand for?

Answer 46

Reverse Transcription Polymerase Chain Reaction ## Footnote RT PCR is a laboratory technique used to amplify RNA.

Question 47

What is the significance of mature RNA?

Answer 47

It is the final form of RNA after splicing ## Footnote Mature RNA is essential for proper gene expression and protein synthesis.

Question 48

True or False: DNA is involved in the process of splicing.

Answer 48

False ## Footnote Splicing occurs at the RNA level, not DNA.