1. a. What was the size of your pGAL plasmid when cut once with Eco R I or Hind

Question

1. a. What was the size of your pGAL plasmid when cut once with EcoR I or Hind III ?

b. Does this match the total size of the pGAL plasmid?

c. What size protein would you expect when this sequence is expressed in E. coli? (assume that the average molecular weight of an amino acid residue is ~110 daltons)

2. Does the estimate for the size of plasmid in Question 1 match the size estimated from the double digestion with EcoR I + Hind III?

3. What is the yield and purity of each plasmid (ng DNA) estimated by nanodrop spectrophotometry?

Nanodrop Results.

            A260/A280: 1.88

            DNA Concentration: 220.2 µg/µL

Lanes 2-5 are the lanes I am concerned about.

Lane 2: no enzyme

Lane 3: Hind III

Lane 4: EcoRI

Lane 5: Hind III + EcoRI

Explanation / Answer

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain, in this case RY13. The last part of its name, the I, denotes that it was the first enzyme isolated from this strain. EcoRI is a restriction enzyme that cleaves DNA double helices into fragments at specific sites.

The enzyme then cuts the backbones of both strands, allowing the DNA to separate into two pieces. For example, the enzyme EcoRI (see the figure, left, top) binds to the recognition sequence GAATTC and cuts between the G and the A. It also cutsbetween the A and the G on the complementary strand.

HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.

For HinD III, the species is Haemophilus influenza, strain D, third isolated enzyme from that strain.

The size of your pGAL plasmid is 4000bp as understood from the ladder and the gel picture given in the question, when cut once with EcoR I or Hind III.

It does not match with the original size of pGAL plasmid as total size of the pGAL plasmid is 6751bp.

Dalton (Da) is an alternate name for the atomic mass unit, and kilodalton (kDa) is 1,000 daltons. Thus a protein with a mass of 64 kDa has a molecular weight of 64,000 grams per mole.

The size of protein to expect when this sequence is expressed in E. coli is 185.185 kDa protein.

The the size of plasmid in Question 1 does not match the size estimated from the double digestion with EcoR I + Hind III as it is 3kb DNA = 111.111kDa protein.

Reliable measurement of DNA concentration and purity is important for many applications in molecular biology. DNA concentration can be assessed using four different methods: absorbance (optical density), agarose gel electrophoresis, fluorescent DNA-binding dyes and a luciferase-pyrophosphorylation-coupled quantitation system. The two most common methods of measuring DNA purity and concentration are absorbance (measured using a spectrophotometer) and agarose gel analysis.

We typically measure DNA purity and concentration using a NanoDrop™ spectrophotometer. A260/280 and A260/230 values greater than 1.8 are typically suitable for analysis. Lower A260/280 values may indicate protein contamination.

The most common technique to determine DNA yield and purity is measurement of absorbance.

DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml

Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (ml)

To evaluate DNA purity, measure absorbance from 230nm to 320nm to detect other possible contaminants. The most common purity calculation is the ratio of the absorbance at 260nm divided by the reading at 280nm. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the DNA unsuitable for any application, but lower ratios indicate more contaminants are present.

The ratio can be calculated after correcting for turbidity (absorbance at 320nm).

DNA purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)

Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230 is best if greater than 1.5. A reading at 320nm will indicate if there is turbidity in the solution, another indication of possible contamination. Therefore, taking a spectrum of readings from 230nm to 320nm is most informative.

1. 1 kb DNA

1. ?

1. 333 amino acids

1. 1.35 kb DNA

1. ?

1. 50 kDa protein

1. 5 kb DNA

1. ?

1. 185.185 kDa protein

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