Dna Extraction

2 February 2017

EDTA tubes: tubes with purple colored caps; called so because they contain EDTA {Ethylene Diamine Tetraacetic acid} * Empty blood sample into falcon tubes-larger tubes that are graduated so lessen the work and can accommodate up to 50mL * Add a solution to dilute to 40 mL without touching to avoid cross contamination * Shake well * Centrifuge for 10 minutes DNA extraction takes place from the nuclei of leukocytes. EDTA is simply an anticoagulant {otherwise if blood clots; DNA extraction will not be possible}.

We rely on EDTA because it doesn’t interfere with later analysis such as diagnostic tests or molecular research.In this experiment we follow a series of steps in order to extract the DNA from the nucleus; this is done by: * Cell lyses using hypotonic solution. Cell lyses solution: mixture of salts whose tonicity is regulated such that it causes rupture of the cell membrane only. It is important to regulate the tonicity because initially; we don’t want to rupture the nuclear membrane. When we rupture the cell membranes {of WBCs}; the nuclei will be released, which can then be collected by centrifugation. * Next step to reach the DNA; is rupturing the nuclear membrane; same way we use nuclei lyses solution.Upon rupturing the nuclear membrane, DNA + RNA + proteins will be released.

Dna Extraction Essay Example

RNA will not cause an issue because it is a very sensitive molecule that will not be able to resist harsh conditions, and is easily degraded. The only problem is with the proteins. * In order to remove proteins; we make use of organic solvents: Phenol Chloroform – which should be used in the ratio 1 : 1 {for eg. If my blood sample is 10 mL; I use 5mL Phenol and 5mL Chloroform}. Phenol is very powerful in disrupting/degrading proteins and chloroform is a very powerful organic solvent. Two phases will be created : * Aqueous phase containing DNA Organic Phase Degraded proteins will assemble at the boundary between the two phases. Using a Pasteur pipette; we transfer the DNA containing phase into a clean tube.

We repeat this step twice. We can aid in this separation by centrifuging for 2-3 minutes under low rpm. This step is a bit stressful; so we have to be very careful while pulling out the aqueous phase, you shouldn’t touch the inter phase otherwise you will be taking the proteins again ! It is a time-consuming step. * The DNA is actually dissolved in the nuclei lyses solution, so we precipitate the DNA by adding a good volume of alcohol; in this case; isopropanol.After centrifugation, pour off the liquid to obtain clean DNA. Once we get rid of 70% ethanol, even if traces of alcohol are still associated with DNA, leave the tube open for some time and all of the alcohol will evaporate; leaving clean DNA. However we cannot preserve DNA in the precipitated form because it will be unstable and will get degraded.

Therefore we usually preserve the DNA in a clean liquid such as double distilled water or de-ionized water but preferentially we use a buffer called TE good preservative for DNA. It is not advisable for students to use phenol chloroform hazardous chemicals.With pharmaceutical advancement; we started to rely less on manual procedures of DNA extraction and we started to rely more on Kits – a collection of liquids. Aim behind the kit is that it provides you with all the reagents needed from the beginning till the end of the procedure while saving time. In this experiment the Kit that we used contained: * Cell lyses solution * Nuclei lyses solution * New solution called “Protein Precipitation solution”- precipitates proteins after nuclei lyses. Instead of using Phenol Chloroform – use this solution and then continue with the normal steps. Rehydrating Solution If you freeze DNA, you will have to thaw it before it is usable.

These cycles of freeze – thaw will degrade the DNA. After dissolving the DNA in the liquid; we take a small amount and store it in the fridge small amount of the large amount, this can be used until you finish your experiments while preserving the original genomic DNA undisturbed. Important Points: * The amount of centrifugal force depends on the speed. The higher the speed, the greater the centrifugal force * The lower the density of the articles; the higher the speed that will need to be used to precipitate those particles. * Vortex machine: helps pellets that are stuck at the bottom of the tube to re-suspend. We use this machine to minimize any losses after centrifugation. Qn.

If we were supposed to centrifuge at 5,000 rpm for 10 minutes, and the maximum I have in the lab is 3,800 rpm. If we increase the time, will this compensate for the difference in rpm? Ans. No, because the amount of speed that I select gives me the amount of force that I need to precipitate the particles.

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