EXAM 1 REVIEW

Multiple Choice

100

What are the steps of PCR?

Denaturing, Annealing, Elongation

200

Does TaqPolymerase have proofreading activity?

No, it is unable to correct errors, and it needs proofreading enzymes to remove mismatched bases

300

Is PCR amplification exponential?

Yes, because both strands are being copied

400

What is RT-PCR used for?

Quantification and measuring gene expression

500

(T/F) Sybr Green Dye binds to ssDNA

False. Binds to all dsDNA (not specific) in the minor groove

Multiple Choice

100

What is an alternative to ethidium bromide (EtBr)?

TaqMan Probe

200

What cleaves the TaqMan probe when the primer is extended?

5’ --> 3’ exonuclease activity of the TaqPolymerase

300

(T/F) Attachment of reporter to probe will increase fluorescence?

False. Fluorescence occurs when reporter is removed from probe via TaqPolymerase exonuclease activity

400

How is cycle number defined?

When fluorescence crosses a threshold of detection

500

(T/F) Lower initial concentration will require more cycles to reach the threshold

True. You have less genetic material to work with and it is harder to amplify  

Short Answer

100

(T/F) Higher initial concentration will require more cycles to reach the threshold

False. There is more product in the beginning and thus you need less cycles to amplify

200

What are some loading controls used in Relative RT-PCR?

Actin and GAPDH

300

Which type of PCR would you use to measure gene expression?

RT-PCR. Endpoint PCR is used for cloning

400

What is the purpose of comparative Ct experiments?

Determine the change of gene expression in a sample when compared to a reference sample (i.e., endogenous control)

500

Define Insertional Inactivation

Insertional inactivation is a selection method where an insert is inserted inside of a functional gene (i.e., lacZ) which then results in disruption of the functionality of the gene product.

Short Answer

100

What is the R/M system? 

Utilization of restriction enzyme on either plasmid or vector will cut that specific site sequence

200

List the first 5 steps in primer desgin when given tissue and a plasmid map with MCS/RE sites

• Extraction of mRNA from tissue
• Use reverse transcriptase to convert mRNA to DNA
• Make forward and reverse primers based on MCS and NEB’s 0-cutter list          
• List specific enzymes used in Forward and Reverse primers
• Run end-point PCR – this is because it’s used for cloning
• Visualize PCR products using gel

300

List the next 5 steps to primer design when given tissue adn plasmid map with MCS/RE sites

• Cut out correct sized band & gel purification
• Elute cDNA in water
• Digest plasmid with the same REs used on insert
• Treat with phosphatase
• Digest the insert ends where RE sites are

400

List the last 5 steps to primer design when given cDNA and plasmid map with MCS/RE sites 

• Use DNA ligase to ligate plasmid with vector
• Transform into competent bacteria add antibiotic onto plate – usually the antibiotic used is Ampicillin and Kanamycin
• Pick colonies & extract DNA
• Do another RE digest
• Run a gel – this is to check if the fragments are the right size

500

What is Blue and White Screening ? List the steps 

• Check if the plasmid has a lacZ operon, gene will insert into MCS of lacZ
• Plasmid needs a LacZ repressor. IPTG will bind to it, IPTG is responsible for the removal of the lac repressor, which will allow the gene of interest to be expressed
• Add X-gal and IPTG to the agar
• + dimethyl formamide to the X-gal (since X-gal is insoluble)
• X-gal is used to see the blue colonies
• Colonies that are white indicate that the plasmid has the gene of interest, since the LacZ gene is disrupted

Short Answer

100

List 2 controls you use when performing a cloning procedure 

• PCR Control:  Positive: control template (checks PCR reagents; visible band expected)
• Negative: Water (ensure sample is free of contamination; no visible band)
• Uncut Vector Control: checks the efficiency of transformation and competency of bacteria or if an incorrect antibiotic was used

200

List 2 controls used when cloning 

• Insert Only Control: tests if plasmid is present in colonies and if there is no contamination (should yield NO colonies)
• Vector and RE Digest Control: Tests if restriction enzyme and digest was successful (should yield NO colonies)

300

List 2 controls used in a cloning procedure.

• Vector, RE Digest, and Phosphatase Control: ensures phosphatase is working. Phosphatase removes the 5’ phosphate so it can’t religate with itself
• Vector, RE Digest, and Ligase Control: tests for the efficiency of the ligase

400

List a control used in cloning procedures 

• Ligase & Restriction Enzyme (from MCS) Control: ensures the ligase can ligate the vector after being cut with restriction enzymes
• *SPECIFIC TO QUESTION 1 ON EXAM 1 PRACTICE PROBLEM SET*