McClean: Colony PCR (Yeast)-PDF

Overview

Our lab’s version of yeast colony PCR, was adapted from the Botstein Lab’s protocol. Generally, we use this protocol for checking transformations (ie, to check that a drug marker or fluorescent protein has been inserted into the genome correctly) or for PCRing up a piece of DNA from the genome to send for sequencing.

Materials

• HotMaster Taq Polymerase
• 10x HotMaster Buffer with Mg²⁺
  • The polymerase and buffer come in the 5 Prime kit FP220320 ordered from Fisher
• 10mM dNTPs
• Forward primer (10μM)
• Reverse primer (10μM)
• Sterile H₂O

Protocol

Add approximately 0.6μL of cells (tiny amount) with the tip of a sterile toothpick into the bottom of the PCR tubes or plate. Once you’ve put cells into the PCR vessel, put the end of the toothpick into ~100μL sterile YPD in either an Eppendorf or the well of a culture plate. You will use this to inoculate an overnight culture if your colony PCR works. Keep the Eppendorf or culture plate at either room temperature or 30°C while you run the PCR, either is fine.

Microwave cells in the PCR tube/plate for 1min (2X). Put microwaved cells on ice.

Add the reaction mix (described below) to the PCR tube/plate. It is recommended to make up a master mix if you are doing multiple colonies. Put the PCR tubes/plate into the thermocycler and run the Colony PCR program described below.

PCR Reaction Mix

PCR Program

Run PCR on Colony PCR program :

• 95°C 4min
• For the following steps, reduce the temperature ‐0.5°C each cycle and cycle 30x’s
  • 94°C 1min
  • 65°C 1min
  • 68°C 2min
• For the following steps, cycle 30x’s
  • 94°C 30sec
  • 50°C 30sec
  • 72°C 1min
• 72°C 5min
• 4°C 5 min
  • Please don’t leave the thermocycler running at 4°C for longer than an hour or so, it wears out the machine. If you need to leave your PCR for longer, please change the last step of the program so that instead of holding at 4°C the program just ends (letting the samples come to room temperature). Letting the sample come to room temperature, even overnight, does not seem to cause any problems for the DNA.

• Taylor D. Scott 20:32, 19 March 2016 (EDT): I had success with the following program adapted from the touchdown protocol and the HotMaster manual (see references below). The amplification is lower (as is expected with fewer cycles), but it might work if you just need to quickly verify a colony.

• 95°C 4 min
• Repeat the following 3094°C 30 sec
  • 52°C 30 sec (use the Applied Biosystems calculator [see references below] to find the right annealing temperature)
  • 65°C 1 min 30 sec (approximately 1 min/kb)
• 72°C 5 min
• 4°C hold

‘Colony PCR with the cheap Taq’

Add approximately 1/4 of a colony to a PCR tube and microwave for 30 sec. Immediately put the tubes on ice after microwaving. To each tube add the following:

Mix by pipetting up and down.

Run PCR with the following program:

• 94°C 5 min
• Repeat the following 25X
  • 94°C 30 sec
  • T_a 30 sec
  • 72°C 1 min/kb
• 72°C 5 min
• 4°C hold

Where T_a is the appropriate annealing temperature for your primers. Gradient PCR can be used if the calculated T_a is not working.

‘Agarose Gel Electrophoresis’

• Find stock gel or prepare the agarose +ethidium bromide gel according to these instructions: McClean: Agarose for gels
  • Ethidium bromide is a known carcinogen. Use gloves, don’t inhale fume, and follow good lab practice.
• Melt the gel in the microwave and let it cool until it is warm but not painfully hot to the touch.
• Fill the gel container with the agarose gel until it just begins to overflow out of the small clear plastic chamber into the large container.
  • 
• Let the gel dry
• Place your gel in the electrophoresis container and ensure that the TAE solution is filled to the marked line. If not, find the TAE solution in the stock room dilute it to 1x solution, and fill the container.
  • The DNA will have a net negative charge, and so ensure that the holes in the gel face the anode (-) so that the DNA will flow to the cathode (+).
  • 
• Insert 10 microL of DNA ladder (your reference points) into a well. For reference, the 1kb ladder mix has 0.5kb, 1kb, 1.5kb, 2kb 3kb (double bright), 4kb, 5kb, 6kb, 8kb, and 10kb fragments of DNA.
• On parafilm, pour ~1 microL drop of “10x blue juice” found in the “Gel Supplies Wisc Mini Fridge.”
  • 
  • Add 10 microL of your PCR product to the drops, pipetting up and down to mix them.
  • Pipet your PCR product blue juice into each well. It is denser than the salt solution and will sink into the hole.
• Press “Set” on the device so that the two LEDs next to “Volt” are lit, and then press the “Run” button. (You may need to set a maximum amount of time too)
• A ~500bp fragment of DNA will require ~45min to be properly separated. A 7kb fragment of DNA will require ~1 1/2 hrs to be distinguishable.
• Place your gel slabs in the UV chamber, turn the UV chamber on, take a picture, turn on the connection to the printer, turn on the printer, and then print the image by selecting Set> Print on the camera.
  • 
• Save the picture in your lab notebook and document the significance of each column.
  • 

‘Freezing Down Positive Transformants’

Once you have run the colony PCR and confirmed which colonies are correct and which are not, take the 100μL of YPD that you set aside before, inoculate it into 4mls of fresh YPD in a test tube, and grow it to saturation overnight. Use this to freeze down glycerol stocks of the strain the following morning.

Notes

• The lengthy touchdown PCR program protocol was devised by Megan in the early days of the McClean lab when we were struggling with getting colony PCR to work reproducibly. The program is probably WAY longer and more complicated than it needs to be (and ties up the thermocycler for 4 hours at a time) so it would be worth someone’s time to figure out a shorter program. Once you do that, please add it as another colony PCR protocol on openwetware and add a note here.
• We probably don’t need to be using the Hotmaster Taq nor do we need to be doing 50μL reactions. If someone has some spare time, please try optimizing the protocol to use our super-cheapy Taq (ask Megan for where the stock is) and do smaller reaction volumes.
• The cheap Taq protocol can probably be scaled back to 50 μL per reaction, but I haven’t tried it.

References

Botstein Lab protocols: http://www.princeton.edu/genomics/botstein/protocols/colony_PCR.htm

HotMaster manual: https://www.5prime.com/media/3388/hotmaster%20taq%20dna%20polymerase%20manual_5prime_1044359_032007.pdf

Applied Biosystems Tm calculator: http://www6.appliedbiosystems.com/support/techtools/calc/