Lab 4: SDS Page Analysis Flashcards
What is the purpose of SDS PAGE analysis?
To confirm protein expression and purification by visualizing expressed proteins and estimating their molecular weight.
What is the composition of the 12% resolving gel for SDS PAGE?
- 30% acrylamide mix (40:1)
- 1.5M Tris.Cl pH 8.8
- 10% SDS
- 10% ammonium persulphate
- TEMED
What is the pH of the Tris buffer used in the stacking gel?
pH 6.8
Fill in the blank: The stacking gel is prepared with _____ acrylamide mix.
30%
What is the volume of the stacking gel prepared in the protocol?
3ml
What is the purpose of overlaying the gel mix with 0.1% SDS solution?
To allow the gel to set properly.
What should be done after the gel has set for at least 20 minutes?
Pour off the 0.1% SDS overlay and mix and add the stacking gel.
What type of buffer is used to fill the inner and outer buffer chambers?
Tris-glycine buffer
At what voltage should the samples be electrophoresed?
100V
What is the purpose of the bromophenol blue dye in the electrophoresis process?
To indicate when the samples have reached the bottom of the gel.
What is the staining solution used after electrophoresis?
1% commassie blue stain
How long should the gel be stained with commassie blue stain?
1 hour
What is done to destain the gel?
Transfer to a solution lacking commassie stain for several hours, changing the buffer after one hour.
What type of disorder is Prader-Willi syndrome?
Imprinting disorder
Caused by failure to express the SNRPN gene product.
Which gene is associated with Prader-Willi syndrome?
SNRPN (small nuclear ribonucleoprotein-associated polypeptide N)
Expressed only on the paternal chromosome 15q11.
How is the maternal SNRPN gene affected in Prader-Willi syndrome?
Methylated and not transcribed
This leads to loss of expression of the SNRPN gene.
What genetic events can lead to Prader-Willi syndrome?
- Deletion of paternal allele
- Maternal isodysomy
Both lead to loss of expression of the SNRPN gene.
What type of disorder is Angelman syndrome?
Imprinting disorder
Caused by failure to express the UBE3A gene product.
Which gene is associated with Angelman syndrome?
UBE3A
Expressed only on the maternal chromosome 15q11.
How is the paternal UBE3A gene affected in Angelman syndrome?
Methylated and not transcribed
This leads to loss of expression of the UBE3A gene.
What genetic events can lead to Angelman syndrome?
- Deletion of maternal allele
- Paternal isodysomy
Both lead to loss of expression of the UBE3A gene.
What technique is used for diagnosis in the provided content?
Southern blotting and methylation sensitive restriction enzymes
This technique is utilized to analyze DNA fragments for genetic disorders.
What is the characteristic of Not 1 enzyme?
Methylation sensitive
Not 1 recognizes and cuts methylated DNA.
What is the characteristic of Xba 1 enzyme?
Methylation insensitive
Xba 1 does not recognize or cut methylated DNA.
What is the size of the DNA fragment obtained from cutting the paternal chromosome 15q11 with Not 1/Xba 1?
0.9kb
This fragment size is indicative of Prader-Willi Syndrome.
What is the size of the DNA fragment obtained from cutting the maternal chromosome 15q11 with Not 1/Xba 1?
4.3kb
This fragment size is associated with Angelman Syndrome.
In the context of Prader-Willi and Angelman syndromes, what does a 0.9Kb fragment indicate?
Prader-Willi Syndrome (PWS)
The presence of the 0.9Kb fragment suggests a paternal deletion.
In the context of Prader-Willi and Angelman syndromes, what does a 4.3Kb fragment indicate?
Angelman Syndrome (AS)
The presence of the 4.3Kb fragment suggests a maternal deletion.
True or False: Cutting the paternal chromosome 15q11 yields a larger fragment than the maternal chromosome when using Not 1/Xba 1.
False
The paternal chromosome yields a smaller fragment (0.9Kb) compared to the maternal (4.3Kb).
Fill in the blank: Cutting maternal chromosome 15q11 with Not 1/Xba 1 yields a _______ fragment.
4.3kb
This fragment size is significant in diagnosing genetic syndromes.
What is Southern Blotting?
A method used to detect specific DNA sequences in DNA samples
It involves transferring DNA fragments from an agarose gel to a nitrocellulose membrane.
What is the purpose of size markers in Southern Blotting?
To determine the size of DNA fragments
Size markers are DNA fragments of known lengths used for comparison.
What type of gel is used in Southern Blotting?
Agarose gel
Agarose gel electrophoresis separates DNA fragments based on size.
What enzyme is typically used to digest DNA in Southern Blotting?
EcoRI
EcoRI is a restriction enzyme that cuts DNA at specific sequences.
What is the process of transferring DNA fragments to nitrocellulose paper called?
Blotting
This step involves the transfer of separated DNA fragments from a gel to a membrane.
What solution is used to separate DNA fragments during the blotting process?
Alkali solution
An alkali solution is used to denature the DNA and facilitate transfer.
What is the role of a labeled DNA probe in Southern Blotting?
To hybridize with complementary DNA bands
Labeled DNA probes are used to detect specific sequences on the membrane.
What technique is used to visualize the labeled DNA probe?
Autoradiography
Autoradiography allows for the detection of radioactive or fluorescent labels in the DNA.
Fill in the blank: The DNA fragments are separated by _______ electrophoresis.
agarose gel
True or False: Southern Blotting can only be used for labeled DNA.
False
Southern Blotting can be performed with unlabeled DNA, but labeling enhances detection.
What is the term for the process that modifies mature RNA?
Splicing
Splicing is a crucial step in RNA processing where introns are removed, and exons are joined together.
What does SNRPN RT PCR indicate when absent?
Diagnostic
The absence of SNRPN in RT PCR can be indicative of certain genetic conditions.
What percentage is referenced in the context of SNRPN RT PCR?
82%
This percentage may refer to the sensitivity or specificity of the SNRPN RT PCR test.
Fill in the blank: _______ is always present in the WASP control.
WASP Control
What type of nucleic acid is associated with SNRPN?
RNA
SNRPN (Small Nuclear Ribonucleoprotein N) is involved in RNA processing.
What does the acronym RT PCR stand for?
Reverse Transcription Polymerase Chain Reaction
RT PCR is a laboratory technique used to amplify RNA.
What is the significance of mature RNA?
It is the final form of RNA after splicing
Mature RNA is essential for proper gene expression and protein synthesis.
True or False: DNA is involved in the process of splicing.
False
Splicing occurs at the RNA level, not DNA.
