Lab 2 Flashcards
(10 cards)
When DNA is in insoluble form in nucleus its calle __ or ___
as a long polynucleotide, or called deoxyribonucleoprotein (DNP)
isolation of molcules from tissues: 3 stpes
homoginization, centerfugation, enrichemtn or target molecule (treat to make target easier to get, like seperate by filtation, electric field, ion stregth) (for this one, dna will dissolve in high salt and proteins will percipitate, centerfuge then resoluble DNA with ethonol)
determin DNA [ ] in dilute solutions wiht? 2
dische diphenylamine reaction (takes advantage of the deoxyribose sugars, reacts with any 2-deoxypentose). UV spec (take advantage of physical shape flat ring)
is the DNA extract clean?
260ab/280ab is it greter then 1.85? Yes then clean
You should note that the DNA present in your “crude” sample is only a small portion of the total mass of material extracted. What is the rest of the material?
The rest of the material is parts of the left over cell that we wen’t cable to successfully separate. This can be residual membrane that wasn’t dissolved, DNP complex, proteins that weren’t precipitated, or leftover nuclei. OR RNA OR SALT
The Feulgen Cytochemical Technique has several steps in the staining process, but two steps in particular (HCl and Schiff’s reagent) are crucial to the success of the
process. Explain what the HCl step does and how this is related to the Schiff’s reagent step.
In the Feulgen Cytochemical Technique, HCL is critical because the tissue preparation, when hydrolyzed in HCL ,will hydrolyze only the deoxyribose sugars of the DNA helix. The colourless leucofuchsin (Schiff’s reagent) can bind to the hydrolyzed sugars. Once bound, the red will return to the reagent again. Since the now red leucofuchsin was only bound to the deoxyribose sugars of DNA, the only red parts on the slide will be DNA. This red stained DNA can then be observed under a microscope.
Briefly explain how each of the following reagents contributed to the isolation of the DNA.
i) 95% ethanol ii) DNA buffer iii) 2.6M NaCl iv) Triton-X
I) The 95% ethanol is added to the DNA solution to re-precipitate the DNA into a fibrous mass. This allows the DNA to be substantial enough to be picked up using a fork.
ii) The DNA buffer is added to the DNA solution to prevent any DNase activity. This is possible because DNA buffer contains sodium citrate, the citrate ions will chelate Mg2+ which is essential for DNase activity.
iii) The 2.6M NaCl is added to the DNA solution to dissolve the DNA since DNA is soluble in such a high salt solution. The DNA will dissolve and the proteins will precipitate. This allows separation of the proteins and DNA.
iv) The Triton-X is added to the DNA solution to dissociate the DNP and dissolve any membrane that was left over in the solution from before. This is possible because Triton-X is a hydrophobic detergent.
The dry weight of DNA (in mg) per gram (wet weight) of wheat germ, as determined using the two methods.
Total isolated crude DNA=0.33g (from 5g of total)
Subsample: 25mg of crude sample, and 25ml (0.01M NaOH)
Subsample: concentration of crude DNA is1mg/ml
For Dische Method:
Average concentration of unknown=0.072mg/ml
(0.072/1.0)x100 = 7.2%
Percent of Crude sample that’s DNA:
(0.072x0.33) = 0.024g of DNA
Therefore of the 5g of starting wheat germ, we extracted 0.024g of DNA
4.8x10-3 grams of DNA per grams of starting wheat germ was extracted.
(0.024x5)x100 = 11.8%
For UV Method:
Average concentration of unknown=0.115mg/ml
(0.115/1.0)x100 = 11.5%
Percent of Crude sample that’s DNA:
(0.115x0.33) = 0.038g of DNA
Therefore of the 5g of starting wheat germ, we extracted 0.038g of DNA.
7.59x10-3 grams of DNA per grams of starting wheat germ was extracted.
intesitry of feulgen
1) Is DNA condensed or dispersed whats that look like
2) degree of hydrolysis
3) nucleolis
1) darker colour(heterochromatin), lighter colour (euchromatic)
2) no hydrolysis is light staining
3) nucleolis unstained due to RNA blocking (RNA never stains)
In (b) above, are the values you obtained the same? If you have variation in the results, suggest where this variation may have arisen. Under the conditions that you did this experiment, which method of DNA quantification, do you think, is the most reliable?
Explain your choice.
The values obtained from the UV method and the dische method were not the same. Variation could have arisen due to slightly hydrolyzed DNA in the DNA molecules during the UV test, which unwinds the DNA and and creates higher absorbable readings (which we found 0.115mg/ml in UV vs. 0.072mg/mL in dische) this is called the hyperchromatic effect. Also any contaminate molecules (proteins, RNA, free nucleotides) in the crude DNA could also absorb light at 260nm and effect the reading. However, our 260/280 ratio was ?????which is over 1.85 so we can assume our DNA solution is reasonably clean and this isn’t the main factor creating the difference. Lastly, human error should always be considered. The dilutions might not have been completely accurate, the readings might have been recorded wrong, etc. The Dische test is by far the most reliable test under these conditions, as theres little interfering material present in the cells to alter the results. It’s straightforward and reliable as compared to the UV test where hyperchoratic effect must be taken into consideration.
