The purpose of this practical is to extract DNA by both Youtube procedure or commonly used laboratory process called salting out method. Usual practice is to use one method, mostly, Youtube technique while comparing with effects of other method. Within each method, fruit samples of strawberry and kiwi fruits are handled with extraction buffer agents and micro-centrifuged and prepared for agarose gel electrophoresis (AGE). Reference DNA ladder is additionally used with fruit samples to compare quality, reliability and quantity of DNA extraction.
The document is a science report that records the effects of the tests and discusses them to identify fine and quantity of sample and additionally reliability of methods used. As samples are tested, observations at each stage can reveal results in terms of quality and consistency of sample preparation, testing, observation and interpretation of results. Observations like impact of differences in sample extraction and methods used for testing can be useful to understand the process and further improve for better results. Inverted light images of test results will be evaluated in reference to intensity of reference sample. Higher intensity bands can be considered as having more quantity of bases of DNA fragments and position of band shows the length of the band.
There are 3 different stages that are common to many DNA extraction procedures:
Agarose gel electrophoresis (AGE).
There are two different methods of DNA extraction Cell and Genes Practical Manual:
Method 1: DNA extraction Youtube method
Method 2: DNA extraction – salting out method.
Method 1 –
DNA is extracted from Strawberry and Kiwi.
Sample fruit is sealed in Ziploc and squished for 3 mins.
10 mL of DNA extraction buffer (1000 mL of DNA extraction buffer contains: 950 mL water, 50 mL dishwashing detergent, 2 teaspoons of table salt) is auto-pipetted into the squished sample and again squished for 2 mins.
The mixture is filtered into a conical flask through a chux cloth and funnel.
Transfer 3mL of sample into 15 mL tube using an auto-pipette and label the tube with sample type and initials and put it on ice.
Pour 10 mL of cold 100% ethanol carefully down the side of the tube to observe that a separate layer is formed on top of the liquid. Leave the tube on ice for at least 10 mins while a white fluffy precipitate of the DNA sample forms between the two liquids.
Collect sufficient DNA precipitate (with a hook) into a labelled 1.5 mL micro-centrifuge tube containing 70% ethanol and place in a micro-centrifuge and spin at 12000 rpm for 1-3 mins.
Take the tube out of the micro-centrifuge and carefully pour off the supernatant into a liquid discard beaker and place the tube upside down on a paper towel and leave it to dry for 5 mins for ethanol to evaporate.
Now, add 100-300 μL of distilled water into the tube and draw the sample up and down for 15 times to mix well. Ensure that DNA sample dissolves in water.
Now, place the tube onto the heat block at 55oC for 5 minutes.
Pipette 10 μL of sample into a micro-centrifuge tube containing 2 μL of 6 × loading dye [loading dye contains: 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue (a visible dye), 0.25% (w/v) xylene cyanol FF (a visible dye)].
Load the 2 log molecular weight marker (DNA ladder) for reference.
Load the sample into agarose gel (agarose gel contains; 1 % agarose, TAE buffer, ethidium bromide (EtBr) approx. 0.2-0.5μg/mL) and note the lane.
Connect the gel to the power-source and run it. When the gel has completed its run, obtain an image of the DNA.
Method 2 –
This is a modified method of Sunnucks and Hales (1996) and the first four steps are conducted the day before the practical commences.
1. 10 mg of fruit tissue is finely chopped using a razor blade and placed into a 1.5 mL micro-centrifuge tube.
2. 10 μL of Proteinase K (an enzyme to break protein) is added to the tube and tube is inverted 3 times.
3. 600 μL of TNES Buffer is added to the sample (TNES buffer contains 50 mM Tris, 400 mM NaCl, 20 mM EDTA & 0.5 % SDS).
4. The sample is mixed and incubated at 37 °C overnight.
5. The sample is labelled with initials and fruit type.
6. Add 170 μL 5M NaCl (a saturated solution) to the sample using an auto-pipette, and vortex (using a bench top of closing the lid of tube with finger and flick to mix) for approximately 10 seconds until the sample precipitates.
7. Place the tube into the micro-centrifuge and spin at 12000 rpm (top speed) for 5 mins as the precipitated protein forms a pellet at the bottom of the tube while the DNA remains dissolved in the solution.
8. Whilst centrifuging is taking place, a clean micro-centrifuge tube is labelled with initials, fruit type and date and later filled with 300 μL of the supernatant (liquid part).
9. Add 300 μL of cold 100% Ethanol to this tube using an auto-pipette and invert (turn upside down) gently 3-4 times. This causes the DNA to precipitate.
Then Incubate at room temperature for 10 mins and micro-centrifuge at 12 000 rpm (full speed) for 5 minutes to pellet the DNA at the bottom of the tube.
Carefully remove the ethanol using an auto-pipette, being extremely careful not to dislodge the tiny pellet (white smear of DNA). Drain any remaining ethanol by placing the micro-centrifuge tube upside down on a piece of paper towel for 2 minutes.
Add 300 μL of cold 70% ethanol to the micro-centrifuge tube and wash the DNA pellet or any remaining protein contamination.
Micro-centrifuge at 12 000 rpm for 5 mins to ensure all the DNA is pelleted at the bottom.
Carefully remove the ethanol, making sure not to touch the pellet (white smear of DNA), using an auto-pipette.
Place open Micro-centrifuge tube onto the heat block at 37°C for 20 minutes to ensure all ethanol evaporates.
Add 20 μL water to Micro-centrifuge tube and mix gently until DNA dissolves.
Pipette 10 μL of sample into the micro-centrifuge tube containing 2 μL of 6 × loading dye [loading dye contains: 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue (a visible dye), 0.25% (w/v) xylene cyanol FF (a visible dye)].
Load the 2 log molecular weight marker (DNA ladder).
Load sample into the agarose gel (agarose gel contains; 1 % agarose, TAE buffer, ethidium bromide (EtBr) approx. 0.2-0.5μg/mL) and make a note of the lane the sample is pipette.
Connect the gel to the power-source and run it. When the gel has completed its run, obtain an image of the DNA as result.
Following are the test results for samples for strawberry and kiwi fruits. Two methods of DNA extraction have been employed and the results are as shown below:
Figure : DNA extraction of sample using Salting Out method.
Figure : Test results for fruit sample using Salting Out method.
Figure 3: DNA extraction of sample using Youtube method.
Figure 4: Test results for sample test using Youtube method.
Figure 5: DNA sample extraction using Youtube method.
Figure 6: Test results for samples using Youtube method.
Figure 7: DNA extraction for using Salting Out method.
Figure 8: Test results for extraction using Salting Out method.
As observed in test results (Fig 2 & 8), samples of DNA extraction have more weight as they are extracted using salting out method. It can be observed that quality of the base pairs of bands of DNA extraction is high for this method. Consistency too is high when compared to reference bands at the different band levels. The length of base band pairs too is high as the distance travelled by them in the different bands is greater. Using salting out method, difference in molecular weight of DNA of both samples is due to quality and consistency of sample preparation methods and efficiency of tester to extract the pellet of fruit. However, quality of base band pairs is not too high but volume is high, when compared to reference.
With Youtube method, we could not find any base band pairs with enough volume and weight to propagate the gel as we could not record any bands, except in one cell. This can be due to the lack of quality in sample preparation as well as inefficiency of method itself. As the presence of base band pairs could reveal size of band pairs, the results in Fig 6 can be evaluated as having one sample with high quantity of DNA (of kiwi) and smears of bands are unclear and indicate that quality is low. Size of bands on all of the results in Fig 2 & 8 show that their base bands correspond to lower and middle intensity reference sample. However, in Fig 6, bands corresponding to all intensities are present in high volumes and have lesser lengths due to which they could propagate the gel to higher intensity base pairs.
It is observed that while the method used to extract DNA sample may not be rightly analyzed using the results, efficiency in preparation can greatly impact size, quality and quantity of sample. Consistency of results can indicate that salting out method is more useful to obtain higher size samples but efficiency of extraction can be improved to improve quality of sample and consistency of results.
SLE111. Cells and Genes Practical Manual. 2017. Deakin University, Australia.
Practical 3 – DNA Extraction. 2017. Deakin University, Australia.