Lab 3: CF Flashcards
What is the purpose of the PCR reaction in cystic fibrosis mutation detection?
To amplify specific DNA sequences for mutation detection
PCR (Polymerase Chain Reaction) is used to increase the quantity of a specific DNA segment.
What are the components added to a 500 ul PCR tube for the reaction?
- 0.2 ul Primer mix (100 ng/ul)
- 17.3 ul H2O
- 5 ul dNTP stock (10 mM)
- 5 ul 5X PCR buffer (7.5 mM Mg)
- 1 ul Taq (5 u/ul)
- 1 ul DNA
Each component has a specific function in the PCR process.
What temperature and duration are used for the initial denaturation step in PCR?
94°C for 3 minutes
This step is critical to separate the DNA strands before amplification.
What is the annealing temperature and duration in the PCR cycle?
59°C for 30 seconds
This is the temperature at which primers bind to the DNA template.
What is the extension temperature and duration in the PCR cycle?
72°C for 30 seconds
This step allows the DNA polymerase to synthesize new DNA strands.
How many cycles are typically performed in this PCR protocol?
35 cycles
The number of cycles can affect the yield of the PCR product.
What is the final extension time in the PCR process?
72°C for 5 minutes
This step ensures that any remaining single-stranded DNA is fully extended.
What is the purpose of electrophoresis in this protocol?
To separate and visualize the amplified PCR products
Electrophoresis is a technique used to separate DNA based on size.
What voltage and duration are used for electrophoresis in this protocol?
150 V for approximately 3.5 hours
This allows the xylene cyanol dye to run off the bottom of the gel.
Fill in the blank: The positions of the primers are found in the _______ sequence.
[CFTR exon 10]
The CFTR gene is associated with cystic fibrosis.
What is the acrylamide concentration used for delta F508 sizing in the miniATTO system?
40% acrylamide (19:1)
This concentration is specifically designed for the miniATTO system.
What buffer is used in the gel preparation for delta F508 sizing?
TBE
TBE stands for Tris-Borate-EDTA, a common buffer used in gel electrophoresis.
What is the purpose of TEMED in gel preparation?
Catalyst for polymerization
TEMED (N,N,N’,N’-tetramethylethylenediamine) is used to initiate the polymerization of acrylamide.
How much APS is used in the gel preparation?
2.5 ml
APS (ammonium persulfate) acts as a radical initiator for the polymerization process.
What is the voltage and duration for running the gel?
150 volts for approximately 3.5 hours
This duration is until xylene cyanol has just run off the bottom of the gel.
What staining method is used to visualize DNA in the gel?
Ethidium bromide staining
Ethidium bromide intercalates with DNA and fluoresces under UV light.
How long should the gel be placed in the ethidium bromide solution for staining?
10 minutes
This allows sufficient time for the ethidium bromide to bind to the DNA.
What is used to visualize the stained DNA?
Transilluminator
A transilluminator provides UV light to visualize the DNA bands in the gel.
Fill in the blank: The amount of TEMED and APS used in the miniATTO system is _______ the amount normally used.
double
This increased amount is necessary for proper well formation in the small ATTO system.
What is the purpose of the Sigma Enhanced Avian RT-PCR kit?
To perform reverse transcription and PCR reactions in a one tube system
This kit simplifies the process by combining reverse transcription and PCR into a single step.
What primer is typically used for AMV reverse transcriptase?
Oligo dT
Oligo dT allows reverse transcription of all poly adenylated mRNAs.
What is the role of the reverse PCR primer in the one tube system?
It provides the primer needed for AMV reverse transcriptase to target specific RNA
This allows for focused amplification of desired RNA sequences.
What temperature and duration are used for the reverse transcription reaction?
48°C for 45 minutes
This step is crucial for converting RNA to complementary DNA.
What components are included in the reaction tube for RT-PCR?
RNA, AMV reverse transcriptase, thermostable AccuTaq DNA polymerase, dNTP, forward and reverse primers, and buffer
These components are necessary for the successful amplification of target sequences.
What gene sequences are amplified in the described RT-PCR reaction?
SNRPN gene and WASP sequences
SNRPN is associated with Prader Willi syndrome.
What is the initial step in the practical schedule for setting up the RT-PCR reaction?
Add components to a 0.5ml tube
This includes preparing a premix of all components except RNA.
What is the function of the RNase inhibitor in the RT-PCR reaction?
To prevent degradation of RNA
This ensures that the RNA template remains intact for reverse transcription.
What is the cycling condition for the final extension in PCR?
7 minutes at 68°C
This allows for the completion of DNA synthesis.
Fill in the blank: The RT-PCR reaction initially performs reverse transcription at _______ for 45 minutes.
48°C
True or False: The AMV reverse transcriptase can function without a primer.
False
A primer is essential to initiate reverse transcription.
What is the function of the thermostable AccuTaq DNA polymerase in the RT-PCR?
To amplify the DNA generated from reverse transcription
This enzyme withstands the high temperatures used in PCR cycling.
What is the purpose of the MgCl in the RT-PCR reaction?
To act as a cofactor for DNA polymerase
Magnesium ions are essential for enzyme activity during PCR.
What is the significance of amplifying both SNRPN and WASP sequences simultaneously?
It allows for multiplex RT-PCR
This technique enables the detection of multiple targets in a single reaction.
