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4.1 Molecular Biology > Lab 4: SDS Page Analysis > Flashcards

Lab 4: SDS Page Analysis Flashcards

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1
Q

What is the purpose of SDS PAGE analysis?

A

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

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2
Q

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

A
  • 30% acrylamide mix (40:1)
  • 1.5M Tris.Cl pH 8.8
  • 10% SDS
  • 10% ammonium persulphate
  • TEMED
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3
Q

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

A

pH 6.8

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4
Q

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

A

30%

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5
Q

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

A

3ml

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6
Q

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

A

To allow the gel to set properly.

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7
Q

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

A

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

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8
Q

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

A

Tris-glycine buffer

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9
Q

At what voltage should the samples be electrophoresed?

A

100V

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10
Q

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

A

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

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11
Q

What is the staining solution used after electrophoresis?

A

1% commassie blue stain

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12
Q

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

A

1 hour

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13
Q

What is done to destain the gel?

A

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

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14
Q

What type of disorder is Prader-Willi syndrome?

A

Imprinting disorder

Caused by failure to express the SNRPN gene product.

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15
Q

Which gene is associated with Prader-Willi syndrome?

A

SNRPN (small nuclear ribonucleoprotein-associated polypeptide N)

Expressed only on the paternal chromosome 15q11.

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16
Q

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

A

Methylated and not transcribed

This leads to loss of expression of the SNRPN gene.

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17
Q

What genetic events can lead to Prader-Willi syndrome?

A
  • Deletion of paternal allele
  • Maternal isodysomy

Both lead to loss of expression of the SNRPN gene.

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18
Q

What type of disorder is Angelman syndrome?

A

Imprinting disorder

Caused by failure to express the UBE3A gene product.

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19
Q

Which gene is associated with Angelman syndrome?

A

UBE3A

Expressed only on the maternal chromosome 15q11.

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20
Q

How is the paternal UBE3A gene affected in Angelman syndrome?

A

Methylated and not transcribed

This leads to loss of expression of the UBE3A gene.

21
Q

What genetic events can lead to Angelman syndrome?

A
  • Deletion of maternal allele
  • Paternal isodysomy

Both lead to loss of expression of the UBE3A gene.

22
Q

What technique is used for diagnosis in the provided content?

A

Southern blotting and methylation sensitive restriction enzymes

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

23
Q

What is the characteristic of Not 1 enzyme?

A

Methylation sensitive

Not 1 recognizes and cuts methylated DNA.

24
Q

What is the characteristic of Xba 1 enzyme?

A

Methylation insensitive

Xba 1 does not recognize or cut methylated DNA.

25
Q

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

A

0.9kb

This fragment size is indicative of Prader-Willi Syndrome.

26
Q

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

A

4.3kb

This fragment size is associated with Angelman Syndrome.

27
Q

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

A

Prader-Willi Syndrome (PWS)

The presence of the 0.9Kb fragment suggests a paternal deletion.

28
Q

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

A

Angelman Syndrome (AS)

The presence of the 4.3Kb fragment suggests a maternal deletion.

29
Q

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

A

False

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

30
Q

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

A

4.3kb

This fragment size is significant in diagnosing genetic syndromes.

31
Q

What is Southern Blotting?

A

A method used to detect specific DNA sequences in DNA samples

It involves transferring DNA fragments from an agarose gel to a nitrocellulose membrane.

32
Q

What is the purpose of size markers in Southern Blotting?

A

To determine the size of DNA fragments

Size markers are DNA fragments of known lengths used for comparison.

33
Q

What type of gel is used in Southern Blotting?

A

Agarose gel

Agarose gel electrophoresis separates DNA fragments based on size.

34
Q

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

A

EcoRI

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

35
Q

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

A

Blotting

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

36
Q

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

A

Alkali solution

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

37
Q

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

A

To hybridize with complementary DNA bands

Labeled DNA probes are used to detect specific sequences on the membrane.

38
Q

What technique is used to visualize the labeled DNA probe?

A

Autoradiography

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

39
Q

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

A

agarose gel

40
Q

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

A

False

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

41
Q

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

A

Splicing

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

42
Q

What does SNRPN RT PCR indicate when absent?

A

Diagnostic

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

43
Q

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

A

82%

This percentage may refer to the sensitivity or specificity of the SNRPN RT PCR test.

44
Q

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

A

WASP Control

45
Q

What type of nucleic acid is associated with SNRPN?

A

RNA

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

46
Q

What does the acronym RT PCR stand for?

A

Reverse Transcription Polymerase Chain Reaction

RT PCR is a laboratory technique used to amplify RNA.

47
Q

What is the significance of mature RNA?

A

It is the final form of RNA after splicing

Mature RNA is essential for proper gene expression and protein synthesis.

48
Q

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

A

False

Splicing occurs at the RNA level, not DNA.

4.1 Molecular Biology (14 decks)

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