Draw your own PCR diagram to show the role of each component and relevance of each temperature shift during the PCR reaction. What’s happening at each of those temperatures and why is it important it changes?
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Draw your own PCR diagram to show the role of each component and relevance of each temperature shift during the PCR reaction.
What’s happening at each of those temperatures and why is it important it changes?
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- 4. Create a PCR master mix for the following: 48 samples, 25 uL total volume per reaction, 2 uL template DNA volume, 2.5mM MgCl, 0.5 µM each primer. Your stock concentrations are: reagent mix (2X, does not contain MgCl,), primers (F/R; 10 uM), MgCl, (50 mM). The master mix calculation must include a 10% overage, and all answers are to be given to one decimal place.Complete this Master Mix table for 8 DNA samples, a positive control, negative control, and an extra reaction for pipetting error. Show your work. Master Mix Conc. Of Total μL µL/Rxn (total per tube) Final Number of Stock Conc. Reactions needed for solution master mix PCR buffer 50X 1X Water DNTP mix 100 mM 200 µM MgCl2 Forward 24 mM 2.5 mM 2 μΜ 0.1 μΜ primer Reverse 2 μΜ 0.1 μΜ primer Taq polymerase DNA 5 U/uL 0.03 U/ µL 1 μL 60 μL Total volume of the entire reaction (µL)In a conventional PCR reaction cycle, which of the following step is NOT needed for a successful PCR reaction? anneal amplification refrigeration O denature
- DNA Extraction by Alkaline Lysis Procedure: 1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip. The spins can be performed at 4C or at room temperature. Longer spins make it difficult to resuspend cells. 2. Resuspend pellet in 100µl GTE solution and let sit 5 min at room temperature. Be sure cells are completely resuspended. 3. Add 200µl NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5 min. 4. Add 150µl potassium acetate solution and vortex at maximum speed for 2s to mix. Place on ice for 5-15 min. Be sure mixing is complete. 5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA. 6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4 ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids. 7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and…Which of the following polymerases should be BEST used if you would want a highly accurate PCR product? high fidelity polymerase hot start polymerase o long amplification polymerase Taq polymeraseDNA Extraction by Alkaline Lysis Procedure: 1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip. The spins can be performed at FC or at room temperature. Longer spins make it difficult to resuspend cells. 2 Resuspend pellet in 100pul GTE solution and let sit 5 min at room temperature. Be sure cells are completely resuspended. 3. Add 200ul NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5 min. 4. Add 150ul potassium acetate solution and vortex at maximum speed for 2s to mix. Place on ice for 5-15 min. Be sure mixing is complete. 5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA 6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4 ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids. 7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and…
- What is the relationship between fluorescence intensity of a spot and the amount of DNA in a sample for a Virochip DNA microassay? From the following which is the best choice Less DNA results in greater fluorescene since the laser passes through he sample eaiser More DNA equals to more intense fluorescence since the DNA makes a layer to refelct the laser light More DNA equals to more intense fluorescence since of more dye on the DNA is fluorescene More DNA results in to more fluorescence since there's more DNA to be excted by the laser None of the abovePrimer annealing is an important aspect of PCR. The annealing step of the cycle usually takes place at around 45-55°C and lasts for only 20-30 seconds. Which of the following statements best explains why the annealing time has to be so brief? OA. A longer annealing time would prevent primers binding to specific sites. OB. The short time frame prevents primers from binding to each other. OC. If the time were any longer, DNA polymerase would begin to denature. OD. The short time frame minimizes complementary template strands base pairing with each other. Reset Selectionat Arrange Window Help 4) 57% D Tue 4 May Tools Slide Show 2 ARMS PCR nations Slide Show Review View A A =、=<|EE|三v| v v A Sha v 20 Quick Styles D Sha Text Arrange Convert to SmartArt Picture Shapes ab x x. AV v Aa v D. Av A、申。 Box Briefly outline how MLPA probe-sets can simultaneously detect multiple targets using a single set of PCR primers. E Notes Comments MAY 4 Pr w MacBook Air
- 2. PCR analysis practice One 1) If you collect samples from crime scene and extract DNA out of each sample. Describe the technologies used to get the below results. 2) Identify which suspect is at the crime scene 3) Among suspect 1-3, who is homozygous for the tested locus and who is heterozygous? DNA from crime scene DNA ladder Suspect DNA #1 #2 #3 500 bp 400 bp 300 bp 200 bp 100 bp | |DNA EXTRACTION Materials: knife 1 cup of fruit Table salt clean piece of cloth or strainer for filtering resealable bag dishwashing liquid 70% rubbing alcohol (chilled) shot glass (or any transparent and narrow container resembling a test tube) How to do the extraction: Before you begin, make sure you have chilled your alcohol in the freezer for at least 3 hours. Room temperature alcohol will not work nearly as well as cold alcohol. Place about 50 mL of alcohol in the freezer to chill it and take it out only when you’re going to need it. (The alcohol will not actually freeze.) 1. The first thing you will need is a sample. Since DNA is found in all living…(a) Describe the actions with reason that should be taken under the following situations: (i) An unlabelled collection tube with a requisition for a V Leiden test is received in the laboratory. (ii) After PCR, the amplification control has failed to yield a product. (iii) DNA is stored overnight in a refrigerator set at 8oC but reads 14 oC. (iv) An isolated DNA sample is to be stored for at least 6 months. (v) The expiration date on a reagent has passed. (b) Explain briefly about genetic testing for new-born, including the importance of genetic testing and the disorder that genetic testing can detect.