qPCR of cDNA_Protocol_Fa2023 - Tagged (1)

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Drexel University *

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Chemistry

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Jan 9, 2024

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Uploaded by ColonelPorcupine794

qPCR of gadA Target Gene and 16S rRNA Reference Gene from cDNA Made from Reverse Transcription of RNA Extracted from Bacterial Cultures Purpose : The purpose of this lab is to run a qPCR on the cDNA created via reverse transcription from the RNA extracted from each bacterial culture. This will quantify, in real time, the amount of transcript made at the time of RNA isolation. Procedure: 1. Label 2 new tubes to be used for the dilution of your cDNA samples. In each tube, you will create a 1:10 dilution of your cDNA sample. Put in 2 µL of your cDNA samples into the appropriate tubes. Add 20 µL of RNAse-free H 2 O into each tube. Vortex for 10 seconds each to mix. 2. Using the Nanodrop, find the concentration of cDNA in each of your diluted samples. Record below: Concentration of A: Concentration of B: 260/280 ratio: 260/280 ratio: 260/230 ratio: 260/230 ratio: 3. Label 4 new 1.5 mL tubes according to which cDNA sample will go in each tube: Tube Contents A1 cDNA from Culture A B1 cDNA from Culture B A2 cDNA from Culture A B2 cDNA from Culture B

4. Create the qPCR reaction mixture by adding in the following reagents to each newly labeled tube according to the table below: **DO NOT ADD IN cDNA TEMPLATE YET!!** Tube 2X QuantiTect SYBR Green PCR Master Mix 16s rRNA Forward Primer (0.3 µM) 16S rRNA Reverse Primer (0.3 µM) gadaA Forward Primer (0.3 µM) gadaA Reverse Primer (0.3 µM) Template cDNA 500ng =____ µL RNase- free H 2 O Total Volume A1 25 µL 1.5 µL 1.5 µL 1.5 µL ___ µL ___ µL 50 µL B1 2 5 µL 1.5 µL 1.5 µL 1.5 µL 1.5 µL ___ µL ___ µL 50 µL A2 25 µL 1.5 µL 1.5 µL 1.5 µL 1.5 µL ___ µL ___ µL 50 µL B2 25 µL 1.5 µL 1.5 µL 1.5 µL 1.5 µL ___ µL ___ µL 50 µL 5. Once all of the tubes are assembled give the tubes a quick spin to pellet all liquids to the bottom of the tube. 6. After the quick spin aliquot in the amount of cDNA as noted in the chart. You need to add the volume (ul) needed to give you 500 ng. Remember that you did a 1:10 dilution before doing the nanodrop. You need to multiple the concentration displayed by the nanodrop by 10 to take into account the original 1:10 dilution. Once you have the volume of cDNA and other reagents added, then determine the amount of RNase-free water needed to give a total volume of 50 ul. 7. Once the cDNA is placed in the tube give it another quick spin to ensure all liquids are mixed at the bottom of the tube. 8. Give your tubes to the TA, who will then plate your samples on the PCR plate. Please note below where on the plate they have placed your samples. (Ex. A1 is in row A, column 2) Record : A1 : row A column ______ A2 : row H column ______ B1 : row A column ______ B2 : row H column ______

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