Screening phage plaques
Adapted from :
When phages successfully propagate among bacteria growing in a lawn, plaques are formed. These are areas of clearing where phages have infected and lysed cells. Plaques can range in size, turbidity, and morphology. Often, it is necessary to collect a sample of the phages that have made a particular plaque (or putative plaque) to perform further experiments with them (such as spot tests or further purification rounds). This protocol describes how to “pick” the plaque and obtain a liquid sample of phage.
1. Distribute 100 ul of phage buffer into 10 different microcentrifuge tubes. Label these tubes 1-10.
2. Place a new sterile pipet tip on the end of your pipet. Pick one plaque on the plate and touch the center of the plaque once with the tip. The idea is to penetrate the top agar layer, but not go all the way through to the bottom agar. The angle of the pipette tip is not critical, although with small plaques, approaching the plaque perpendicularly seems to work best.
3. Move the tip to the prepared tube of Phage Buffer, lower the point under the level of the liquid, and shake moderately from side to side to release phages into the buffer. It’s also a good idea to pipet up and down several times to “wash” any phages from inside the tip into the buffer.
4. Repeat for tubes 2-10, picking a fresh plaque for each tube. Allow to sit at room temperature for 1 hour.
5. Parafilm your bacterial plates from last week and store in the fridge until we analyze the PCR reactions.
PCR of Phage Plaques
Now that we have selected several plaques and isolated the phages, we need to check whether these phages have the synthetic Gene 68 or the native Gene 68. The synthetic Gene 68 contains two watermark sequences within the synthetic gene which mark the DNA sequence as being synthetic. We will therefore perform PCR with primers that bind to these watermark sequences. If the synthetic sequence is present, the primers will bind and begin the amplification reaction and we will get a DNA product. If the primers do not bind because the synthetic sequence is absent then we will not get a DNA product amplified.
When screening, we want to start with several plaques to maximize the likelihood that we have at least one that is correct (contains the synthetic gene). We will therefore be setting up 12 PCR reactions (10 different plaques plus positive and negative controls).
1. In a large microcentrifuge tube on ice, combine the following components:
•140 ul of 2X master mix (2X MM) •28 ul of primer mix •104 ul of sterile water •8 ul of DMSO
2. Pipet up and down 10X to completely mix the contents of the tube.
3. Aliquot (distribute) 20 ul per tube of the mix into 12 PCR tubes (the very small ones).
4. Into PCR tube 1, add 1 ul from phage tube 1.
5. Repeat step #4 for phage tubes #2-10.
6. Into PCR tube #11, add 1 ul from tube PC.
7. Into PCR tube #12, add 1 ul of sterile water.
8. Put your 12 PCR tubes into one column of the PCR machine and begin the PCR reaction.
98°C, 30 seconds
98oC, 10 seconds
55oC, 30 seconds
72oC, 30 seconds
72oC, 10 minutes
9. If the PCR reactions indicate that we have successfully introduced the synthetic gene 68 into the rest of the phage genome, we now have a stock of the phage sample (the plaque that you picked into phage buffer). If none are positive, we can pick more plaques and screen more phages for positives.
Pouring a Gel:
1. Weigh out 0.5 g of agarose on a piece of weigh paper. Transfer to an Erlenmeyer flask. Add 50 ml of 1X TAE.
2. Place the flask in the microwave and heat until the agarose is completely transparent and colorless.
3. Allow the agarose to cool -this will take about 10 min.
4. While the agarose is cooling, place the gel tray into the gel box and add the comb.
5. When the agarose is cool, add 5 ul of Gel Red to the melted agarose
6. Swirl the agarose to incorporate the Gel Red and pour the agarose into the gel tray.
7. Allow the gel to solidify for 15-20 minutes. During this time, you prepare your samples (below).
Preparing your samples:
1. Gather your colony screening PCR products. They should be:
1. Plaque 1
2. Plaque 2
3. Plaque 3
4. Plaque 4
5. Plaque 5
6. Plaque 6
7. Plaque 7
8. Plaque 8
9. Plaque 9
10. Plaque 10
11. Positive control
12. Negative control
2. To each tube, add 4 ul of 6X DNA loading dye.
Running a Gel:
1. Into lanes 1-12, load 11 ul of each of your PCR products (mixed with water and dye).
2. Into lane 13, load 10 ul of the DNA ladder (premixed with water and dye)
3. Place the lid with electrodes onto the gel box, and set voltage to 100V.
4. Run gel approximately 30 minutes or until the dye is 2/3 of the way down the gel, then take a picture.
5. When done, you want to check that:
• There are no DNA bands in the NC lane of the gel (lane 12)
• There is a DNA band in the PC lane of the gel (lane 11).
• Whether or not there are DNA bands in any of your plaque samples (lanes 1-10)