holy-moly havent posted in a long time..almost a week o.o i think...
well.. alot has happened. i am officially fully-fledged in the midst of my 'stressfull period of assignments.' hbio test, chem quiz 3 and the mbc lab report are done with.. now just gotta do my archaeology paper due for friday, and then mbc MID SEMESTER EXAM next week T_T annndd then hbio essay as well. oh and there is also chem quiz number FOUR. so yeaah, lots of stuffs to do.. however, after the end of aug (when my hbio essay is due) its gonna be nice and calm with basically no assignments.. just the usual ongoing study. and then the mid sem break will come which will be uber joyous.
but seriously cannot wait till this month is over. im happy just thinking about it lol. last night i was up till about 1:30 am (this morning) doing my mbc lab report. its amazing how much effort we put into something which only has a weighting of like 2% haha. hmm since im bored i shall post it here hahaa. i cbf uploading all the appendices and figures and stuff so its just the report part. (ill post it below)
hmm what else.. oh AWKWARD situation today. i was walking across the uni today and i saw these two ppls i know (lets call them A & B) together.. who were supposedly going out.. soo.. i saw these two ppls together so i texted my friend with a message saying something like this: just saw Person A and Person B sitting together on the oval.. NAAAWWWWW LOL.
i then preceded to recieve a text from my friend which said.. ohh they were breaking up.i was like O.O so it was rather awkward.. im just glad i didnt go upto person A and B when they were sitting 'together' to say hi. THAT would have been BAD.
well i think thats all for nows... cant really think of much else..i shall post my lab report as promised:
Extraction and Quantification of DNA from a Chicken Liver Cell Homogenate
Introduction
All living cells contain deoxyribonucleic acid (DNA) as it is used to store genetic information within the cell (Alberts et.al.2008). All cells are enclosed by a plasma membrane, and within this cell membrane, proteins are embedded (Alberts et.al.2008). Thus, by disrupting this membrane and solubilising the proteins, the DNA can be isolated and extracted. To quantify DNA, many different methods exist, such as using UV spectrophotometry, light spectrophotometry and the wet-weight to dry-weight ratio. A spectrometer is an instrument for producing and examining spectra (Turner 1987). Thus a spectrophotometer is one which does so within or near the visible spectrum. This instrument can then be used to determine the concentration of DNA present within a sample. The wet-weight/dry-weight method is used as there is a known ratio which exists between the weight of ‘wet’ and ‘dry’ DNA, thus the initial concentration of dry DNA can be calculated if the ‘wet’ weight of the DNA is obtained, and the initial volume of the sample is provided.(Appendix A).
The first objective of this experiment was to extract DNA from a chicken liver cell homogenate by exploiting the various solubility properties of the cellular macromolecules. After doing this, the objective was to then quantify the DNA using three different methods; using wet-weight/dry-weight calculations, UV spectrophotometry, and using a light spectrophotometer after treating the DNA sample with diphenylamine.
Materials and Methods
The experiments were carried out as written in the SCIE1106 unit manual (pages23-30). A deviation from the procedure was made in step 8 of laboratory 2, whereby the tubes were cooled in an ice bath for ten minutes, as opposed to being left at room temperature to cool down.
Results
The results for the experiments were as follows:
After centrifugation in step 5 of Lab 1, three separate layers were observed; a bottom organic layer, a middle protein layer, and a upper aqueous layer which contained the dissolved DNA. Upon extraction of the uppermost layer in step 7 of Lab 1, 3.5 ml’s of the aqueous layer was collected without disturbing the underlying protein layer. In step 8 of Lab 1, after the ethanol was carefully added to the 3ml of aqueous DNA solution which was isolated in the previous steps, two distinct layers were observed. The ethanol was added by using a Pasteur pipette to introduce the ethanol down the side of the tube, such that it could flow down onto the aqueous DNA layer. In step 9 of Lab 1, the DNA became visible when the two layers were mixed, thus it could be wound onto the end of the Pasteur pipette. After the DNA was left to drain, the wet weight of the DNA was 29.00mg. This wet weight was converted to a dry weight as shown by the calculation in Appendix A. The dry weight of the DNA was 1.827mg. This dry weight of DNA was then used to calculate the concentration of DNA present in the liver-cell homogenate. A concentration of 4.035mg dry DNA/g of liver was obtained. The calculations are attached as Appendix B. An UV spectrophotometer was also used to quantify the concentration of the DNA and RNA in the solution, along with the concentration of proteins. The readings are as follows:
260nm=0.0672 (concentration of DNA, RNA and other free nucleotides)
280nm=0.315 (concentration of proteins)
In laboratory 2, the DNA was treated with diphenylamine, and a standard curve produced. This curve is included as Figure 1. The table from which this standard curve was produced is attached as Table 1. Upon consultation of the graph, the ½ dilution and undiluted DNA values were observed as follows:
½ dilution: ~0.015mg in 500μl
Undiluted: ~0.030mg in 500μl
As the amount of DNA in the ½ dilution corresponded to approximately half of the DNA present in the undiluted sample, the initial pipetting and dilutions were observed as being accurate. Using the standard curve, the concentration of DNA in the initial liver cell homogenate was calculated to be 0.33125mg dry DNA/g of liver. The calculations are attached as Appendix C.
Discussion
As the results of these experiments demonstrate, the primary objectives to extract DNA from a chicken liver cell homogenate and then quantify it using the three different methods were met. However, the three different quantification methods resulted in three very different DNA concentration values due to the different nature of the quantification methods. In the quantification method in Laboratory 1 (in which the wet-weight/dry-weight ratio was used) the ‘wet-weight’ of the DNA would have also included ribonucleic acid (RNA) and other free nucleotides, thus contributing to a heavier wet-weight. Furthermore, as the ‘wet’ DNA was only left to drain off the excess ethanol for ten minutes, a large quantity of ethanol would still have been present when the wet DNA was weighed, thus also contributing to this inaccuracy. As this wet-weight served as the basis for further calculations, this resulted in the significant difference in the concentration values obtained.
The three different methods of DNA quantification which were used in these experiments each had their own advantages and disadvantages. Whilst the wet-weight/dry-weight method was a quick and easy method, it had many disadvantages, as discussed above. In addition to these, the use of a lab scale meant that only very large quantities of DNA could be quantified, as the weight of the DNA must have been large enough to register on the scale. Likewise, the UV spectrophotometry method is a quick and simple method; however the absorbance at 260nm also measured the concentration of RNA and other nucleotides. In addition, an UV spectrophotometer is expensive. The final method used in these experiments was the use of diphenylamine reagent, which also had its merits. As diphenylamine reacts solely with the deoxyribose sugar, the spectrophotometer detected only the DNA, and not other nucleotides; thus giving the most accurate quantification. Furthermore, as the diphenylamine reacted within the visible spectrum, a cheap light spectrophotometer was all that was required. A disadvantage for this method was the labour-intensive, time consuming procedure in which precise pipetting was required when creating the microtitre plate. In addition, the handling of hot acids and use of other dangerous chemicals could have lead to a laboratory hazard if precautionary steps were not adhered to.
The use of UV and light spectrophotometry has been used to quantify DNA in other experiments, such as the one explained within the journal article ‘Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA’ (Shokere, Holden & Jenkins 2009). As explained within the article, DNA was quantified using a spectrophotometry machine at 260nm, and by using flourometry. The results from this experiment concluded that the flourometry method provided a more accurate quantification of the DNA; similarly in our laboratory experiments, it was concluded that the diphenylamine method was the most accurate method of quantification. In accordance to the experiment explained within this article and many similar experiments in other journal articles, our results are reflective of the ones which have been obtained within these experiments, thus seem to be valid.
In conclusion, the diphenylamine method of quantification was regarded as being the most accurate method due to the use of the deoxyribose-specific reagent. The A260nm method and the wet-weight/dry-weight method were considered inaccurate as the quantified amount included the concentration of RNA and other free nucleotides. In addition, the presence of ethanol in the latter method lead to further inaccuracies when calculating the concentration from the wet weight. As our results and conclusions were consistent with other scientific literature, the experiments’ results were regarded as legitimate.
