M465:Colony PCR

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PCR Amplification of 16s rRNA genes from Universal Bacterial Primers

Last time we performed PCR we were using DNA extracted from a kit as a template. Today you will use a lysate, made from your isolated colonies, instead.

Creating a cell suspension as a template for PCR

1. For each of your isolates, obtain a tiny 0.2 mL PCR tube filled with 50 uL of distilled water. Label each tube with your isolate designation. For example, if I was working with isolate 3, I might label my tube with my initials (IN) such that the tube would be labeled: IN-3. This should match what is written on your agar plate.
2. Using a sterile toothpick, resuspend a well isolated colony from your plates in the 50 uL of water.
3. Take your PCR tube to your instructor. She will place these at 98C in the PCR machine for 10 minutes.
4. After the incubation is complete, use your lysates in the PCR reactions below.

Protocol for PCR
For each of your isolates, obtain a tiny 0.2ml pcr tube from your instructor. All of the ingredients listed below in the table, except the template, have been added together previously and kept on ice for you in these tubes. Don't forget to label your tubes!

1. To each tube, you will add 2 μL of the cell suspension/lysate. Since your pcr tube already has 10μL master mix, 6μL DNAase free water, and 1μL of each of 2 primers, the total reaction volume for everyone will be 20μL.

It is very important to pipet these tiny volumes accurately. Use the P10 or P20 pipettes. Look at the tip after you draw up your measured volume to make sure you have liquid there.

2. Dispense the lysate into the liquid directly, watching to make sure that the liquid has left the pipette tip.

3. Bring your tube to your instructor; they will show you where the thermal cycler is located in JH 022. Keep track of where in the PCR machine your tubes have been placed (the exact quadrant, row and column). Your instructor will start the reaction when everyone's tubes are loaded.

Component TABLE

Component amt. in a 20 μl
reaction
Final Conc.
Purified
DNAase free
Water
6 μL already in tube.
Want to achieve
total of 20 μl reaction vol.
Add from 0 - 3μl
_
2x Phusion Master Mix 10 μl 1x
16S 27F primer 1 0.5 μMolar
16S 1492R primer 1 0.5 μMolar
template lysate 2 μl optimum is 100ng of DNA/reaction


The cycling program is shown below.


94°C for 2 min 30 cycles at: 94°C for 30 s 56°C for 45 s 72°C for 1 min 30 s 1 cycle at: 72°C for 10 min

4°C hold