Lab 3: CF

Question 1

What is the purpose of the PCR reaction in cystic fibrosis mutation detection?

Answer 1

To amplify specific DNA sequences for mutation detection

PCR (Polymerase Chain Reaction) is used to increase the quantity of a specific DNA segment.

Question 2

What are the components added to a 500 ul PCR tube for the reaction?

Answer 2

• 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.

Question 3

What temperature and duration are used for the initial denaturation step in PCR?

Answer 3

94°C for 3 minutes

This step is critical to separate the DNA strands before amplification.

Question 4

What is the annealing temperature and duration in the PCR cycle?

Answer 4

59°C for 30 seconds

This is the temperature at which primers bind to the DNA template.

Question 5

What is the extension temperature and duration in the PCR cycle?

Answer 5

72°C for 30 seconds

This step allows the DNA polymerase to synthesize new DNA strands.

Question 6

How many cycles are typically performed in this PCR protocol?

Answer 6

35 cycles

The number of cycles can affect the yield of the PCR product.

Question 7

What is the final extension time in the PCR process?

Answer 7

72°C for 5 minutes

This step ensures that any remaining single-stranded DNA is fully extended.

Question 8

What is the purpose of electrophoresis in this protocol?

Answer 8

To separate and visualize the amplified PCR products

Electrophoresis is a technique used to separate DNA based on size.

Question 9

What voltage and duration are used for electrophoresis in this protocol?

Answer 9

150 V for approximately 3.5 hours

This allows the xylene cyanol dye to run off the bottom of the gel.

Question 10

Fill in the blank: The positions of the primers are found in the _______ sequence.

Answer 10

[CFTR exon 10]

The CFTR gene is associated with cystic fibrosis.

Question 11

What is the acrylamide concentration used for delta F508 sizing in the miniATTO system?

Answer 11

40% acrylamide (19:1)

This concentration is specifically designed for the miniATTO system.

Question 12

What buffer is used in the gel preparation for delta F508 sizing?

Answer 12

TBE

TBE stands for Tris-Borate-EDTA, a common buffer used in gel electrophoresis.

Question 13

What is the purpose of TEMED in gel preparation?

Answer 13

Catalyst for polymerization

TEMED (N,N,N’,N’-tetramethylethylenediamine) is used to initiate the polymerization of acrylamide.

Question 14

How much APS is used in the gel preparation?

Answer 14

2.5 ml

APS (ammonium persulfate) acts as a radical initiator for the polymerization process.

Question 15

What is the voltage and duration for running the gel?

Answer 15

150 volts for approximately 3.5 hours

This duration is until xylene cyanol has just run off the bottom of the gel.

Question 16

What staining method is used to visualize DNA in the gel?

Answer 16

Ethidium bromide staining

Ethidium bromide intercalates with DNA and fluoresces under UV light.

Question 17

How long should the gel be placed in the ethidium bromide solution for staining?

Answer 17

10 minutes

This allows sufficient time for the ethidium bromide to bind to the DNA.

Question 18

What is used to visualize the stained DNA?

Answer 18

Transilluminator

A transilluminator provides UV light to visualize the DNA bands in the gel.

Question 19

Fill in the blank: The amount of TEMED and APS used in the miniATTO system is _______ the amount normally used.

Answer 19

double

This increased amount is necessary for proper well formation in the small ATTO system.

Question 20

What is the purpose of the Sigma Enhanced Avian RT-PCR kit?

Answer 20

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.

Question 21

What primer is typically used for AMV reverse transcriptase?

Answer 21

Oligo dT

Oligo dT allows reverse transcription of all poly adenylated mRNAs.

Question 22

What is the role of the reverse PCR primer in the one tube system?

Answer 22

It provides the primer needed for AMV reverse transcriptase to target specific RNA

This allows for focused amplification of desired RNA sequences.

Question 23

What temperature and duration are used for the reverse transcription reaction?

Answer 23

48°C for 45 minutes

This step is crucial for converting RNA to complementary DNA.

Question 24

What components are included in the reaction tube for RT-PCR?

Answer 24

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.

Question 25

What gene sequences are amplified in the described RT-PCR reaction?

Answer 25

SNRPN gene and WASP sequences

SNRPN is associated with Prader Willi syndrome.

Question 26

What is the initial step in the practical schedule for setting up the RT-PCR reaction?

Answer 26

Add components to a 0.5ml tube

This includes preparing a premix of all components except RNA.

Question 27

What is the function of the RNase inhibitor in the RT-PCR reaction?

Answer 27

To prevent degradation of RNA

This ensures that the RNA template remains intact for reverse transcription.

Question 28

What is the cycling condition for the final extension in PCR?

Answer 28

7 minutes at 68°C

This allows for the completion of DNA synthesis.

Question 29

Fill in the blank: The RT-PCR reaction initially performs reverse transcription at _______ for 45 minutes.

Answer 29

48°C

Question 30

True or False: The AMV reverse transcriptase can function without a primer.

Answer 30

False

A primer is essential to initiate reverse transcription.

Question 31

What is the function of the thermostable AccuTaq DNA polymerase in the RT-PCR?

Answer 31

To amplify the DNA generated from reverse transcription

This enzyme withstands the high temperatures used in PCR cycling.

Question 32

What is the purpose of the MgCl in the RT-PCR reaction?

Answer 32

To act as a cofactor for DNA polymerase

Magnesium ions are essential for enzyme activity during PCR.

Question 33

What is the significance of amplifying both SNRPN and WASP sequences simultaneously?

Answer 33

It allows for multiplex RT-PCR

This technique enables the detection of multiple targets in a single reaction.