PCR-Gels Exercise

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CUNY LaGuardia Community College *

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Biology

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May 23, 2024

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Uploaded by BrigadierOpossumMaster252

General Microbiology Laboratory – BIOL201 All submissions will be on Blackboard. However, I prefer you submit online AND bring a printed version to class (it is easier for me to grade). Charts/Tables and all work may be hand-written. Polymerase Chain Reaction (PCR) & Gel Electrophoresis Lab Exercise DNA can be found in many different places in the arthropod (Figure 1). All DNA, including nuclear and mitochondrial DNA, from the arthropod, was extracted and purified; additionally, all bacterial DNA was extracted and purified. In this activity, we will use Polymerase Chain Reaction (PCR) to amplify segments of the extracted DNA in order to (i) obtain enough DNA for arthropod identification and (ii) determine whether or not the arthropod is infected with Wolbachia (a genus of bacteria). We will do this by targeting two specific genes: CO1 from the arthropod and 16S rRNA from Wolbachia . After DNA extraction and Purification, a nanodrop spectrophotometer was used to quantify or measure the concentration, and purity of our extracted DNA samples. Two samples were used. [μg/mL] Reading Wavelength Abs. Wavelength Abs. Wavelength Abs. Ratio (230/260) Ratio (260/280) DNA Sample A 230 260 280 DNA Sample B 230 260 280 Sample A Sample B

1. What is the importance of a 230/260 ratio? What does your calculated 230/260 ratio tell you about your DNA samples? 2. What is the importance of a 2 6 0/2 8 0 ratio? What does your calculated 2 6 0/2 8 0 ratio tell you about your DNA samples? After quantifying your DNA samples, you want to prepare PCR tubes to make copies of your DNA samples. 3. Calculate a “PCR Cocktail”, containing forwar d and reverse primers, Taq Master Mix, and water. Imagine that you have 9 samples. To make sure you have enough PCR Cocktails for all of your reactions, make a PCR Cocktail for 10 reactions. 4. Once you aliquot 23 μL of PCR Cocktail into a tube, you would then add 2μL of template DNA for a total reaction volume of ________ μL in each tube.

5. How many controls will you have for each PCR reaction? Describe the purpose of each control. 6. Below is a single strand of DNA. Apply base pairing rules to determine the nucleotide sequence of the complementary strand. 7. What are the expected results if your arthropod is NOT infected with Wolbachia ? ( Hint: Which DNA will be amplified?) 8. What are some things that could cause a PCR to fail? 9. Why are there forward and reverse primers? 10. What would happen if you forgot to use the negative PCR control? 11. How do you think this lab activity is similar to the PCR diagnostic test for COVID-19?

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After your PCR, you run your samples on a 1% agarose gels. 12. Assume that single PCR reactions were loaded into two separate gels for arthropod and Wolbachia DNA analysis. Fill /Draw in the expected bands for lanes 2-7 using the table below. You may use outside resources. 13. In gel electrophoresis, pore size depends on agarose content. Higher % gels have smaller pores whereas lower % gels have larger pores. In this lab, you used a 1% agarose gel. What would happen if you used a 2% agarose gel, but ran it for the same amount of time? Continue to the next page. Lane Sample 1 DNA Ladder (bands already shown) 2 An arthropod sample positive for Wolbachia 3 An arthropod sample negative for Wolbachia 4 (+) Arthropod Control 5 (-) Arthropod Control 6 (+) DNA Control 7 Water Arthropod (COI) gel Wolbachia (16S rRNA) gel Arthropod Barcoding Gene (CO1): 708bp Wolbachia Specific Gene (16S rRNA): 438bp Expected Band Sizes

14. Based on these results, which step of the experiment likely went wrong? Explain. Are the arthropods tested here confidently infected with Wolbachia? 15. Based on these results, which step of the experiment likely went wrong? Explain. Are the arthropods tested here confidently uninfected? Lane Sample 1 DNA Ladder 2 Sample A 3 Sample B 4 (+) Arthropod Control 5 (-) Arthropod Control 6 (+) DNA Control 7 Water Lane Sample 1 DNA Ladder 2 Sample A 3 Sample B 4 (+) Arthropod Control 5 (-) Arthropod Control 6 (+) DNA Control 7 Water

How to Interpret Gel Electrophoresis Results To interpret gel electrophoresis results, first ensure that all controls are correct. The DNA ladder, (+) Arthropod control, (-) Arthropod control, and (+) DNA control should produce bands of expected size, whereas the water lane should be empty. 1. DNA Ladder To accurately read the gel, confirm the band size of experimental samples by comparing their location in the gel to reference bands in the DNA Ladder. Refer to the information sheet accompanying your DNA ladder for specific band sizes as the bands, or rungs, vary by product. Below is the DNA Marker (M3104) from MiniOne. 2. Positive Controls The (+) Arthropod control and (+) DNA control should have both the CO1 and Wolbachia band present. In a duplex PCR, as shown below, these will appear in the same lane. In a standard (single) PCR, these will be loaded into separate gels. The DNA ladder bands should be clearly present and separated. 3. Negative Controls The (-) Arthropod control should have a CO1 band, but no Wolbachia band. The negative water control should not have any band or smudge. If all controls worked, the results of your experiment are valid, and the experimental bands can be analyzed. If the controls have unexpected results, or if there is a band in the water lane, refer to the Troubleshooting guide on page 16. 1000bp 500bp 2000bp 250bp 100bp Lane 4 Lane Sample 1 DNA Ladder 2 (+) Arthropod 3 (-) Arthropod 4 (+) DNA 5 Water Loading Key Primer Dimers Loading Well Ladder 3 2 5 1 Band

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