1-12(photo) 13. Add 0.25mL SDS to each tune. Cap and vortex for 30 seconds. What
ID: 135376 • Letter: 1
Question
1-12(photo) 13. Add 0.25mL SDS to each tune. Cap and vortex for 30 seconds. What is the purpose of this 14. Incubate the test tubes in the water bath at 70-75 degrees Celsius for 10 minutes. This step helps to disrupt the membranes and dissociate any proteins from DNA. What does heat do to proteins? 16. Incubate on ice for 10 minutes. Ammonium acetates will precipitate the proteins. Why do we need to do this? . Microcentrifuge Ethyl alcohol(95-100%), pre-chilled Water bath (37°C and 70-75?) Microcentrifuge tubes (1.5 mL) . Razor blades . Plastic Petri dishes . Ice buckets . Distilled water Rack for 1.5 mL tubes in the freezer Small plastic pestles " Safe Imager Blue-light Transilluminator Basic Procedure Work in groups of 4. Make sure at least one person in your group is willing to kill the beetles and extract the soft tissues. Part I: DNA extraction 1. a) If you are using larvae, you should use 2-6 larvae, depending on the size of the larvae. If the larvae are too small, you may use 6 or more; ask your instructor. Place 1 larva (or more than 1, if the larvae are small) in each of 2 plastic Petri plates on ice Move to step 2. b) If you are using adult bean beetles you will need to use 10 adult bean beetles per tube. If you are using adult beetles of another species, your instructor will tell you how many adults to use. Place the adults in a microcentrifuge tube. Move to step 7 2. Cut the head of each larva using a razor blade Squeeze one larva from tail to head in order to force out the soft tissue. If the larvae are small and you need more than one per tube, repeat for the other larvae. You may use forceps to do this. 4. Collect all of the soft tissue in a 1.5 mL microcentrifuge tube and place on ice. 5. Repeat steps 2-4 for the other larva(e) and collect into another 1.5 mL tube. 6. Discard the exoskeletons as instructed. 7 Add 0.25 ml, of the DNA isolation buffer to each microcentrifuge tube containing the :soft tissues from larvae, or the adult bean beetles. Grind with a small pestle for 3 minutes and then vigorously pipette several times (up and down) with a transfer pipette re difn ur ch cap the tubes and 9. Add 1 mL of the DNA isolation buffer to each tube, cap the tubes and invert to mix 10. Centrifuge the tubesfor 30 seconds. Make sure the microcentrifuge is balanced. 11. Decant and discard the liquid (as instructed). Then add 0.5 mL DNA isolation buffer to the pellets in each tube. 12. Grind again with 'the pestle for 5 minutes and vigorously pipette several times (up down) with a transfer pipette for 5 more minutes. 13. Add 0.25 mL SDS to each tube. Cap and vortex for 30 seconds. 14, Incubaté' the tubes in the water bath at 70-75°C for 10 minutes. This step helps to disrupt the membranes and dissociate any proteins from the DNA. What does heat to proteins?Explanation / Answer
Hi Answer:
The step 1-6 are general steps during the DNA isolation process from the beetles or other small insects having an exoskeleton. In these steps, the soft tissues are isolated from the exoskeleton.
In steps 7-12 the grinding of the soft tissue is carried out to capture the cells to extract out the intracellular material such as protein, DNA, and RNA etc. In these steps, DNA isolation buffer is used to stabilize the pH during the cell lysis and DNA extraction because DNA is pH sensitive in nature and any change in pH may damage the DNA of cells during its extraction so to avoid this DNA extraction buffer is used.
In Step 13 0.25 mL of SDS is used. SDS [sodium dodecyl sulfate] is the strong anionic detergent which can solubilize the lipids which are present in the cell membrane. When we add SDS to the extraction mixture it helps to absorb these components of cell membrane from the mixture.
In step 14 the mixture was heated at 70-75 degree to dissociate the proteins from DNA and this high temperature denature these proteins and avoid any harmful effect of these proteins to the cell DNA.
In step 15 ammonium acetate precipitation was carried out at low temperature as we know that the ammonium acetate is used for the precipitation of proteins and the proteins separated and denatured from step 14 can be easily precipitated and these precipitates can be easily removed from the mixture.
These all steps are used to remove the lipids, proteins present in the cell and cell membranes which are come together with DNA during the crushing or rupturing the cells. By these steps, these unwanted lipids and proteins are removed and finally, we got pure DNA.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.