BIO 4442 Exam 1: Molecular Biology Labs 1–4

How do you convert 0.5 mL to µL?

0.5 mL is equal to 500 µL.

Convert 2,750 µL to mL.

2,750 µL is equal to 2.75 mL.

What is the conversion of 0.06 mL to µL?

0.06 mL is equal to 60 µL.

How do you convert 0.4 M to mM?

0.4 M is equal to 400 mM.

Convert 2 mM to µM.

2 mM is equal to 2000 µM.

How do you convert 250 ng/µL to µg/mL?

250 ng/µL is equal to 250 µg/mL.

Which pipette would you use for 7 µL?

Use a pipette suitable for 10 µL.

Which pipette would you use for 14 µL?

Use a pipette suitable for 20 µL.

Which pipette would you use for 100 µL?

Use a pipette suitable for 100 µL.

Which pipette would you use for 450 µL?

Use a pipette suitable for 1000 µL.

How much of 1 M Tris-HCl is needed for a 1,000 µL buffer solution at 20 mM?

20 µL of 1 M Tris-HCl is needed.

How much of 500 mM EDTA is needed for a 1,000 µL buffer solution at 1 mM?

2 µL of 500 mM EDTA is needed.

How much of 5 M NaCl is needed for a 1,000 µL buffer solution at 0.2 M?

40 µL of 5 M NaCl is needed.

How much of 1 M Tris-HCl is needed for a 500 µL buffer solution at 100 mM?

50 µL of 1 M Tris-HCl is needed.

How much of 500 mM EDTA is needed for a 500 µL buffer solution at 5 mM?

5 µL of 500 mM EDTA is needed.

How much of 5 M NaCl is needed for a 500 µL buffer solution at 0.8 M?

80 µL of 5 M NaCl is needed.

How much of 1 M Tris-HCl is needed for a 750 µL buffer solution at 15 mM?

11.25 µL of 1 M Tris-HCl is needed.

How much of 500 mM EDTA is needed for a 750 µL buffer solution at 3 mM?

4.5 µL of 500 mM EDTA is needed.

How much of 5 M NaCl is needed for a 750 µL buffer solution at 0.3 M?

45 µL of 5 M NaCl is needed.

How do you prepare a 100 µM stock from 41.7 nmol (in micro liters)?

How would you make 100 micro liters of a 10 micro molar working stock from this?

You need 417 µL of buffer; Mix 10 µL of 100 µM stock with 90 µL of buffer.

Why do we report centrifugation speeds in rcf rather than rpm?

rcf accounts for the radius of the rotor, providing a more accurate measure of the force applied. RPM only does rotational speed.

What gene and what is the expected size range of that gene that we are targeting?

Trying to amplify an SSU 18S rRNA gene. Gene is between 1800 and 2500 bp.

What is achieved by PCR?

PCR amplifies specific DNA sequences.

List the basic components needed for a PCR reaction.

DNA template, forward and reverse primers, taq dna polymerase, divalent cations, nucleotides.

How did using Thermus aquaticus polymerase improve PCR?

It is heat-stable, allowing for higher temperatures without denaturing. T. aquaticus is from hot tempered areas!

Taq is ___, adding 1000bp/min

fast

How can you amplify a gene of interest from a tube of DNA from multiple organisms?

By designing primers with specific nucleotides for the target gene.

what can you look at when designing primers for an unknown target gene?

genetic homology in similar organisms that could have conserved nucleotides close to our target gene.

What challenge arises when amplifying genes from closely related organisms?

Primers may bind to similar sequences, making it difficult to amplify the specific target.

the forward primer looks like

the same as the top strand 5’ —> 3’

the reverse primer complements

the target gene to strand

to get the reverse complement, you _______ the target gene, then _____

reverse (just switch it around), complement

master mixes help prevent

pipetting error

What occurs at 95°C for 2 minutes in PCR?

Initial denaturation of the DNA template.

What occurs at 95 C for 30s in PCR?

denaturation

What occurs at 50°C for 30 seconds in PCR?

Annealing of primers to the template DNA.

What occurs at 72°C for 2.5 minutes in PCR?

Extension of the new DNA strands by DNA polymerase. Elongation

why do you repeat denaturation, annealing, and elongation 33 times

to exponentially amplify the target sequence

what happens during 72C for 5 min in PCR?

final elongation, finishing

which temperature would you adjust depending on the primer’s melting point?

you would adjust the annealing temperature — so the 50C. make it lower than the melting point

if there’s a longer gene, what would you adjust?

the time for elongation, not temperature

why does nested pcr produce a shorter region?

nested PCRs bind to sequences within the original target gene.

What charge does DNA migrate towards in gel electrophoresis?

DNA migrates towards the positive charge (anode) due to its negative charge.

agarose creates a matrix of linear _______ strands made of repeating units of ____

polymer, disaccharides.

the agarose slows the migration to be

measured

in the agarose, the pH acts as a _____ and the salts help with _____

buffer, charge

a flourescent dye called______ helps to be able to visualize it under blue light

GelGreen

Why is nested PCR performed?

increase the specificity and sensitivity of DNA amplification

the nested PCR makes it to where ____ sequences aren’t amplified

nonspecific

if the primary pcr only amplifies a small amount, the nested pcr

amplifies that to give us more!

nested pcr can give us a ____ band during electrophoresis

clearer

What is the maximum volume range of a P-200 micropipette?

20 to 200 µL

What are the three sizes of tips used for micropipettes?

Small tips for P-2 and P-10, medium tips for P-20 and P-200, large tips for P-1000

What are the final concentrations in a 1X PCR solution after dilution?

1.5mM MgCl2, 200µM dNTPs, 25U/mL Taq DNA Polymerase, and 1X Buffer.

What is the concentration of the working stocks of primers used in PCR?

10µM working stocks.

What is the role of MgCl2 in PCR?

It acts as a cofactor for DNA polymerase during DNA synthesis.

What is the purpose of the Tris-Acetate buffer in gel electrophoresis?

To maintain the pH and provide salts necessary for the charge gradient.

What is the purpose of adding a DNA ladder in gel electrophoresis?

To provide a size reference for determining the length of DNA fragments in the samples.

What is the purpose of the incubation period after plating the cells?

To allow colonies to grow and be assessed for the presence of the insert.