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This is an easy PCR protocol for 10ul reactions.

HOW DOES PCR WORK? Please, please, please know this if you are using this technique. Start HERE https://www.dnalc.org/resources/animations/pcr.html

1. 1M NaOH
2. Taq DNA Polymerase Kit
3. 8 Well PCR Strips
4. 1.5 mL Microcentrifuge Tubes
5. milliq_water
6. DMSO

1. Aliquot 10uL (per well) of 0.02M NaOH into PCR tubes (our stock is usually 1M NaOH so you will need to do a dilution).
2. Using a sterile Pipette Tip, pick a small colony and resuspend in NaOH. Remember to NEVER take a whole colony. Circle the colony that you picked and write your initials and the date.
3. If the solution is cloudy, you've added enough cells.
4. Mark your boil PCR strips with sharpie over the entire top of the strip, and your PCR reaction strips only on one side or in some way that is distinguishable from the boiled strips so they don't get mixed up.
5. Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minute.To do this on our machines, click OPEN METHOD → Yeast boil → Next → Start
6. After removing your boiled strips you will want to spin down the strips for about 1-2 seconds. You can use the small centrifuge on Christine's bench. PLEASE balance your samples.
7. In the mean time, prepare the master mix for the PCR reaction.
8. The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer.
9. We use APEX PCR Kit. Prepare the Master Mix as follows:

For 1 Reaction (tip: add the largest volumes first):

• 4.1ul dH20
• 1uL 10X PCR Buffer
• 3ul 5M Betaine
• 0.2uL dNTPs (10mM each)
• 0.2uL foward primer (100uM)
• 0.2uL reverse primer (100uM)
• 0.1uL Taq

Note:If you are doing a large PCR with multiple primers, you may want to make a large master mix (excluding the primers) and then separate into smaller reaction mixes and add the primers to their respective tubes. MAKE SURE YOU FLICK THE TUBES BEFORE ALIQUOTING TO PCR TUBES OR THEY WILL NOT BE MIXED AND YOUR PCR WILL NOT WORK.

1. Aliquot 9uL of the master mix solution into fresh PCR tubes.
2. Transfer 1uL of boiled samples to the master mix aliquots. Pull from the supernatant of your boiled samples, being careful not to pipette any of the yeast from the bottom.
3. Run the PCR cycle. To do this on our thermalcyclers, you will press Open Method → Colony PCR. Our machines have three different temperature zones. To edit them, you will press Edit → Manage steps → Advanced Options → VeriFlex → And then you will click on the pencil under the annealing step. Be sure to set the temperature to the LOWER Tm of your primer set.

NOTE: If you are doing a PCR of a strain that is deleted for KanMX the approximate bp of the marker is 1600, so the minimum Elongation time should be 2 minutes!