McClean: Colony PCR (Yeast)

Overview

Our lab's version of yeast colony PCR, 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 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 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 eppendorfs 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

Reagent	Volume
10x HotMaster Taq Buffer with Mg2+	5μL
10mM dNTP mix	1μL
10μM Primer 1	1μL
10μM Primer 2	1μL
HotMaster Taq DNA polymerase	0.5μL
Sterile Water	40.5μL
Total Volume	50μL

PCR Program

Run PCR on Colony PCR program :

• 95°C 4min
• For the following steps, reduce the temperature ‐1°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 Forever
  • 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.

'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, 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 TAE solution in the stock room and 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, 1kb ladder mix has 500kb, 1000kb, 2000kb, 3000kb, 4000kb, 5000kb, 6000kb, 7000kb, 8000kb and 9000kb fragments of DNA.
• On parafilm, pour ~1 microL drops 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.
• A ~500kb fragment of DNA will require ~45min to be properly separated. A 7,000kb 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, then print the image by selecting Set> Print on the camera.
  • 
• Save the picture in you 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

Please feel free to post comments, questions, or improvements to this protocol. Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

*Megan N McClean 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.

*Megan N McClean We probaby 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 doing smaller reaction volumes.

*Megan N McClean 15:36, 25 March 2013 (EDT) See Sequencing Colony PCR Product for how I prep my colony PCR products for sequencing

References

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

Contact

Megan N McClean

or instead, discuss this protocol.