1. We use Molarity, and Molar solutions in lab a great deal. From my PowerPoint

Question

1. We use Molarity, and Molar solutions in lab a great deal. From my PowerPoint slides and your previous course work, you no doubt know that: 1M = 1mole/L.

a. But what exactly is a ‘mole’? More to the point, what is it equal to?

b. Furthermore, which has the greater number of atoms, 1 mole of glucose or 1 mole of carbon? i. In 1-2 sentences, BRIEFLY explain your answer.

2. (10pts) Plasmid DNA is isolated in this protocol, but generally not the chromosomal DNA of the Bacteria. In 1-3 sentences offer a reasonable explanation as to why (Hint: review pp429-430).

3. (10pts) What specifically can happen to the plasmid DNA that you isolate when the TE buffer pH is too low? (Hint: review pp429-430)

4. (10pts) What happens if you allow you DNA pellet to dry for too long (Hint: Use Google to find an educational resource and cite it below!)?

5. (10pts) In 1-3 sentences explain why it’s important to remove the ethanol from your mini-prep tubes before adding the TE buffer? In other words, what effect would alcohol contamination from the mini-prep have on a restriction digest? Hint: go to this website and read (cut and paste this into your browser): www.neb.com/tools-and-resources/usage-guidelines/star-activity

6. (20pts) Next week we will digest the plasmid DNA that we isolated in this lab (Lab 8A). Let’s consider how restrictive enzymes work. Look the two samples of DNA shown below:

DNA Sample #1 5’– T C C A G T G A T C T C G A A T T C G C T A G T A A C G T T C G A T 3’– A G G T C A C T A G A G C T T A A G C G A T C A T T G C A A G C T A DNA Sample #2 5’– T C A T C G A A T T C C T G G A A T C A G C A G A A T T C G C A T A 3’– A G T A G C T T A A G G A C C T T A G T C G T C T T A A G C G T A T

If both samples are treated with the restriction enzyme EcoRI [recognition sequence G’AATTC] then indicate the number of fragments and the size of each fragment from each sample of DNA. List the fragment sizes in order of largest to smallest. Sample # 1 Sample # 2 # of fragments:________ # of fragments:________ Sizes of fragments (large small): Sizes of fragments (large small):

7. (20pts) Read the protocol for Lab 8B. a. What is supercoiled DNA

b. Why would it migrate further into an agarose gel if it’s uncut plasmid DNA?

c. What causes this type of DNA to form? d. Is this likely to occur to our isolated plasmid DNA?

Explanation / Answer

ANSWER 1

Answer 2. Plasmid isolation is carried out by alkaline lysis. When we add NaOH in the suspended pellet of cells NaOH breaks H-bonds between the bases of Chromosomal DNA and plasmid DNA. Thus converting dsDNA into ssDNA. When we add neutralization buffer containing potassium acetate decrease the alkalinity of the solution. In alkaline condition, ssDNA starts forming H-bond between the strand. It is easy for Plasmid ssDNA to renature due to its small size but impossible for chromosomal DNA to renature.

answer 3. At very low pH phosphodiester bond present in the DNA break down. If we dissolve plasmid DNA in TE buffer of too low pH plasmid DNA will be sheared.

Answer4. If we dry DNA pellet too long then our DNA won’t be able to dissolve as it will make clump upon adding TE buffer

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