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Approximately 28 drops of liquid, from a medicine dropper or disposable pipette, equals 1mL. Each drop represents what part of one mL?

0.035714286 mL

The dyes that you separated using gel electrophoresis were: Orange G (yellow), Bromophenol blue (purple) and Xylene cyanole (blue). What electrical charge did these dyes carry?

The dyes are negative.

What evidence allowed you to arrive at this conclusion?

The dyes moved towards the positive side (red electrodes). [Opposites attract].

Molecular size can play a role in separation with small molecules moving through the gel matrix more rapidly than larger molecules. The formula (or molecular) weights for these dyes are Orange G (452.38), Bromophenol blue (669.98) and Xylene cyanole (538.62). From your results, did it appear that these molecules were separated clearly on the basis of size?

No, because bromophenol blue was the heaviest but it moved farther than xylene cyanole.

What other factors may have played a role in the separation of these dyes?

Degree of negative charge of the molecule and the shape of the molecule.

Which tube contained a single dye? A, B or C?

Tube C

Name this dye.

Xylene Cyanole

When aspirating a solution, why is it important to actually see the solution enter the pipette tip?

Otherwise the pipette will suck up air.

After loading your gel, did any solution remain in tubes A, B or C?

Yes.

What could account for solution remaining in these tubes?

Pipetting errors.

What is the recognition sequence for BamH I?

GGATCC

In a 5' 3' direction, what sequence of bases represents the "sticky-ends for each?"

(BamHI) GATC and (HindIII) AGCT.

pARA digestion will yield fragments and will be base pairs in length.

2; 4018

pKAN-R digestion will yield fragments and will be bp and bp in length.

2; 4706; 702

Assume you were given a culture of bacteria carrying one or both of these plasmids. Design a simple experiment that you could use to determine which of these plasmids, pARA or pKAN-R, the bacteria in the culture were carrying.

Put ampillician into both culture plates. If the bacteria grows, then the plate contains pARA. If it doesn't grow, it does not contain pARA.

Put kanamycin in both culture plates. If the bacteria grows, the plate contains pKAN-r. If the bacteria dies, the plate does not contain pKAN-r.

Why was it important to place the A+ and K+ tubes in the 70°C water bath before setting up the ligation reaction?

To denature the restriction enzymes. [weaken/destroy]

What do you think might have happened if this step was omitted?

Then the restriction enzymes would've cut out the DNA.

Make a diagram to show how the following sticky ends would join together. (: = hydrogen bonding) See page 3.2 for base pairing example.

DRAW DIAGRAM.

A-T
A-T
G-C
C-G
A-T
A-T

Something like that? :/

Although many recombinant plasmids are possible, draw three possible recombinant plasmids. Include as one of the three the combination in which we are most interested—the one that combines pARA with the pKAN-R fragment carrying the rfp gene.

DRAW.

Could two rfp fragments join together and circularize in the Ligase tube?

Yes, because plasmids can recreate recombinant DNA.

In the DNA molecule, there are two kinds of chemical bonds: covalent chemical bonds and hydrogen bonds. Briefly describe how these bonds differ in strength and where, in the DNA molecule, you would find them.

Hydrogen bonds bond in between nucleotide groups. Covalent bonds bond between the sugar and phosphate groups. Covalent bonds are found at the back of the molecule. Covalent bonds are stronger than Hydrogen bonds.

During ligation, which of the bonds (hydrogen or covalent) form first? Where do they form? Which bonds form next and where do they form?

Hydrogen bonds are first. Hydrogen bonds form between the nucleotide base groups. Then covalent bonds are formed between the sugar and phosphate groups.

DNA ligase is required to form which bond?

Covalent bonds.

Use the following table to compare how your actual transformation results differed
from your predicted results. See page 5.5 for "predicted" results.

DRAW.

If your actual results differed from your expected, propose some reasons that might explain these differences.

My results and predictions were the same.

How many red colonies were present on your LB/amp/ara plate.

5 colonies.

Why did the red colonies only appear on this plate and not the LB/amp plate?

Because the LB/amp plate did not have arabinose (sugar).

Would you expect that some of the bacteria on the LB/amp plate were transformed with pARA-R? Briefly describe how you might test your answer.

Yes. You can inject ampicillin to the LB/amp plates and those that were transformed by the pARA-R
will destroy the ampicillin remaining in the plate.

Approximately how many μL are there in one drop of liquid?

35.7 uL

Four hairs, plucked from your head, will yield about 1 μg of DNA. How many μg of
DNA can be extracted from a single hair root? (Show your work.)

0.25 ug

How many nanograms does this represent? (Show your work.)

250 nanograms.

Draw two circles, one representing pARA and the other pKAN-R. Indicate the positions of BamH I and Hind III restriction sites and other genes. Briefly describe the function of each of these genes. ampr, kanr, araC and rfp.

DRAW.

Amp-r contains the gene for ampillician resistance. Kan-r contains the gene for kanamycin resistance.
AraC is a protein that helps the bacterium make proteins.
Rfp carries the gene for mutated fluorescent protein.

Which antibiotic resistant gene, is found in pARA? In pKAN-R?

Amp-r is found in pARA.
Kan-r is found in pKAN-R.

One of these plasmids has been engineered to express a protein if the gene for that protein is inserted into a specific location. Which of the plasmids has been engineered for protein expression?

pARA.

From what organism did the gene for mutant Fluorescent Protein come?

Sea anemone (Discosoma sp)

Briefly outline, in your own words, the steps required to set-up the plasmid restriction digest.

You need the plasmid map (P-bad) to identify the location of the restriction enzymes. Then the restriction sites will make out the spot where the enzymes are cut out.

Why do you suppose you are asked to set up two tubes without BamH I and Hind III?

... That's a good question Mr. Collo.

Briefly define the term "recombinant DNA."

Recombinant DNA is newly recombinant plasmids. (four restriction fragments being combined to make a new molecule)

What does the term ligation indicate?

To bind together.

In order for two sticky ends to join together, what relationship needs to exist between
them?

The sticky ends need to be complementary.

What properties of the restriction fragments produced in Lab 2 are needed to permit ligation of these fragments?

They need to have the same restriction enzymes so they can have the same sticky ends when cut.

What is the importance step 1 in this protocol?

To denature the restriction enzymes so it doesn't cut out the plasmid when ligated.

Draw three possible recombinant plasmids resulting from pARA and pKAN-R fragments. Include any genes these fragments carry.

DRAW.

Besides using electrophoresis to separate DNA fragments according to their sizes, it can be used to estimate the actual size, in base pairs, of each fragment. For example, we might be looking for a gene and we suspect it is of a certain size; electrophoresis can be used to locate fragments in that size range. In order to do this, we would need to run a gel with a mixture of DNA fragments of known sizes. This mixture called a "marker" or "ladder," serves as a control or a standard to which we can compare the positions of other DNA bands in the same gel.
In the diagram, below, the "marker" lane contains 10 DNA bands of known sizes. These sizes are given below. Using this information and the plasmid maps of pARA and pKAN-R, predict the positions of DNA bands produced the pARA -, pARA +, pKAN-R-, pKAN-R + samples. Hint: first determine how many fragments should appear in each sample, and then determine the size(s) of each fragment. We will omit the "LIG" sample.

DRAW.

Beginning with the restriction digest of pARA and pKAN-R, briefly describe the steps required for constructing your recombinant plasmid from pARA and pKAN-R restriction fragments. Explain your answer including the manipulations of these two plasmids and their restriction fragments.

Put pARA and pKAN-r with restriction enzymes.
Then add A+ and K+to the DNA ligse, buffer.
Then add H20 to A+ and K+.
Then add restriction enzymes to plasmids so they can cut them up leaving sticky ends.

What is "transformation?"

Transformation is the process of taking foreign DNA into a bacterial cell.

What does it mean if cells are "competent"?

It means the cell is ready to receive plasmids.

How would you know if a bacterium gets transformed with a plasmid containing the ampr gene?

If ampillician is present, then the plasmid could start to produce proteins that would destroy the ampillician.

What would be the size of the fragment carrying this gene?

4018 bp.

From what plasmid did this fragment originate?

pARA.

How is the P + culture treated differently from the P - culture?

P+ culture has plasmids added to it.

What is the purpose of the P- culture?

P- culture is the plasmid group control.

Why is it important that the cells you collect from your mouth are mixed with Chelex?

Chelex beads will bind divalent magnesium ions. removing the magnesium ions, the degradtion of genomic DNA by nucleuses is reduced.

What is the purpose of boiling the cell sample?

To lyse the cells and to free the DNA within it.

What does PCR accomplish?

PCR can amplify a piece of DNA and make copies.

The "primers" that are used in this PCR are unique to what locus (location or DNA segment)?

Intron of the TPA gene.

What is an "Alu element?"

An "alu element" are DNA fragments of about 300 bp that is distributed in our genomes.

How many base pairs is the Alu element?

300 base pairs (bp).

Does everyone carry an Alu element in the region that we are amplifying by PCR? Explain.

No, since the locus has two forms. One with 300 bp DNA fragment and one that does not.

What do all of the cells growing on the LB/amp and LB/amp/ara plates have in common?

Amp-r.

What single restriction fragment must they all contain to grow on plates with ampicillin?

Amp-r.

Would you expect that all of the cells growing on the LB/amp/ara plate were transformed with the same plasmid? Explain.

No, because there is no way to tell which fragments DNA recombine so it could be the result of many other factors (amp resistance and kanamycin resistance).

How might you determine which of the cells on the LB/amp/ara plate contain pARA-R, the recombinant plasmid that you've made by ligating the rfp gene with the large pARA restriction fragment?

Looking for the red colonies. The red colonies picked up the rfp gene with pARA-R. (rfp gene = red)

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