Bio Lab Exam 1

What volumes does the P20 micropipettor measure?

1-20 uL

What volumes does the P200 micropipettor measure?

21-200 uL

What volumes does the P1000 micropipettor measure?

201-1000 uL

Why does the double-stranded template DNA have to be separated into single strands (denatured) for the PCR procedure to work?

The single-stranded DNA allows for primers to bind to the strand, enabling DNA polymerase to work

If you wanted to find something else about your genes what would you change in the PCR process?

Type of primer

What did we do in Week 1?

1. Isolate DNA from cheek cell

2. Amplify the region of DNA with A & C, using PCR

What did we do in Week 2?

1. Digest DNA with restriction enzyme - cut C

What did we do in Week 3?

1. Agarose gel electrophoresis to determine if restriction enzyme cut copied DNA or not

Can you reuse primers?

NO

The primers we are using are how many base pairs long?

22

How base pairs long is the DNA sequence that we are amplifying?

919

Will the restriction enzyme (ApaI) cut if the person has a genotype of AA? What is the fragment size?

NO → 919

Will the restriction enzyme (ApaI) cut if the person has a genotype of CA? What is the fragment size?

YES (cuts it in half) → 919 from A allele // 713 & 206 from C allele

Will the restriction enzyme (ApaI) cut if the person has a genotype of CC? What is the fragment size?

YES → 206 and 713

SNP (single nucleotide polymorphism)

A DNA sequence variation that occurs when a single nucleotide in the genome sequence is altered

What could the SNP be at the region we’re looking at?

A or C

What genotype causes a faster metabolism for caffeine?

AA

Amplify

making numerous copies of a segment of DNA

True or False: PCR copies all of the DNA in a sample

FALSE

It copies only a specific sequence of the genetic code from a template DNA, targeted by PCR primers

What are the 3 steps to PCR?

1. Denaturation

2. Annealing

3. Extension/elongation

What is the denaturation step in the PCR process? And what temperature is this done at?

The double-stranded template DNA is denatured by heating, typically to 95°C, to separate the double stranded DNA

What is the annealing step in the PCR process? And what temperature is this done at?

The reaction is rapidly cooled to an annealing temperature of roughly 50-60°C

What is the extension/elongation step in the PCR process? And what temperature is this done at?

The reaction is heated to roughly 72°C for efficient DNA synthesis by DNA polymerase

What type of bond are you breaking when denaturating the DNA strand?

Hydrogen bond

What would happen if the ethidium bromide were not put into the electrophoresis trays?

You would see no bands on the gel

What does ethidium bromide do?

It attaches to DNA & glows pink under UV light

What would happen if the restriction enzyme were really old and non-functional?

All of the agarose gel electrophoreses would show that the DNA is 919 base pairs long because none of them would have gotten cut

• It would show that everyone’s genotype was AA

How does contamination affect this experiment?

The results would show everyone heterozygous (CA genotype) since all of the alleles were mixed

What does the BLAST computer program do?

It compares the DNA sequence entered to known DNA sequences & finds similarities between them

Would you expect bacteria colonies to be different sizes when you grow them at room temperature or at 30 degrees?

The bacteria colonies would be larger at higher degrees because the molecules move faster

Which micropipettor would you use for 650 uL?

p 1,000 uL

How would 650 uL be entered into the micropipettor?

065

What type of bonds are formed between the primers and the complementary DNA bases?

hydrogen bonds

How many bands would show up on the gel if you had a genotype of AA?

1 band

How many bands would show up on the gel if you had a genotype of CC?

2 bands

How many bands would show up on the gel if you had a genotype of CA?

3 bands

When would you use a chi-square test?

If you want to ask whether the frequencies of individuals in specific categories are significantly different than the frequencies you would expect to find based on a hypothesis

• appropriate to use for a categorical variable

When would you use a two-sample t-test?

If you want to ask whether values of a numeric variable measured for two groups are significantly different → allows you test the null hypothesis that the mean value of numeric dependent variable is the SAME for the two

Why does the double stranded template DNA have to be separated into single strands for PCR to work?

So the primers can attach - they can’t attach if they the two strands are attached & the DNA wouldn’t be able to replicate

Why does the PCR produce the correct DNA even when your DNA is contaminated with bacterial DNA?

The primers used in PCR are specific for binding to human DNA

Why are primers more likely to anneal to the template DNA at 42-65 degrees rather than 95 or 72?

42-65 degrees is closest to human body temperature, which is ideal for the protein to work

What would the negative control produce on the gel?

No bands would be present

What is a nutrient agar plate?

A petri dish that contains agar, a jello-like material produced from red algae, that provides the bacteria with nutrients

In a bacterial colony, are the cells genetically identical?

YES

Name one other way to identify a bacterial species

Use their DNA sequences