Hi everyone,
I'm trying to perform colony PCR on a S. cerevisiae industrial strain using Zymolyase. So far, I've used 0.05 - 0.1 U/ml Zymolyase in PBS pH 7.4 per colony, incubate at 37 C for 30 - 60 min, vortex, then heat at 95 C for 10. Then use 1 - 2 ul of lysate per PCR reaction. Colonies are fresh. The templates are both genomic and plasmid-based and range from 600 bp to 4 kb. Occasionally it worked, usually it doesn't and I can't seem to figure out the critical factor. There are times when I can visually see cell debris from lysis, but PCR still produced no result.
I read somewhere that the Zymolyase protocol only worked for very short fragment. Is this true? Could anyone help me troubleshoot this?
Thank you
Hi Thu Tran,
as you mentioned the colonies need to be fresh (after 1 day of incubation) In case of colony PCRs the less you add the better. The more cell debris you add to your reaction the more potential inhibitors are transferred to the reaction. You also need to design your primers carefully. Never use products longer than 500bp. The shorter the better. I used to barely touch a colony with a tip and rub it against the pcr tube and then I added 20microL of MM.
good luck,
Mina
Its been a while since I did yeast colony PCR. Back then I added acid washed Glassbeads 425-600 um to yeast and vortexed them 2x3 min to mechanically open then up.
I agree with Mina Bashir, the less colony material probably the better. As for bacterial colony PCR I used to scrape off a colony, transfer into 50uL water, boil for 5 min, and use 1 uL as template for PCR. Another way would be, also as Mina suggested, barely touch the colony and transfer only a tiny amount into the PCR strip.
Check your primer TMs, and I am sure you know the polymerase can roughly amplify 1kb/minute, so your extension time would have to be about 4 min for a 4kb fragment. Make sure you have enough polymerase (may want to double up) in your mastermix as the polymerase degrades over time. You could do a second PCR using a microliter of the first PCR as template using a fresh mastermix with fresh polymerase. I have noticed that with these second PCR I can often detect the fragments better that are of interest.
Good luck!
Hi Thu Tram,
I are going to send you "one" yeast colony PCR protocol (in pdf), that I always use. For me, it is quickly and perfect. I hope it will be OK also for you.
Good luck¡
Hi,
1200bp. But because the primers were already done. I recommend you shorter than 500bp, I agree with Mina Bashir. But if it is impossible or very difficult to do that primers, at least less than 1Kb.
Regards¡
Hi,
an easier way to perform yeast colony PCR is to add a little bit of the colony in the PCR tube (a trace on the side of the tube is enough), put the sample 2 min in a microwave oven, and add the PCR mix straight away.
The key is to not introduce too much of yeast in the tube, as Mina stated, to prevent inhibition of your reaction. A good quality Taq Polymerase is required.
With that technique, we routinely amplify up to 2.5 kb fragments
To my experience, there're lots of strain specific differences when it comes to colony PCR. Indeed I have also just added a tiny amount of yeast with a 10ul tip directly into the PCR mixture (and spotted the yeasts on a plate), and it mostly works for lab yeast. I was working with an aggregating strain and none of the alternatives worked, so I had to lyse with PCI and glass beads. I had a colleague who could microwave and get products. I have never spun down the lysate as it is not always convenient with PCR tubes. And finally don't forget that sometimes you just ended up with target that is difficult to amplify. In this case adding some Mg2+ or 5% DMSO might work.
Cheers,
I did a transformation of an expression plasmid into a fission yeast strain, and I tried to test if my colonies have got the plasmid, so I searched online and found a lot of useful suggestions on Researchgate. I modified the protocols and made my own protocol. And it worked well enough for my screening (I tested 9 colonies and confirmed 4 of them. The negative ones are not necessarily not transformed though).
1. Prepare a 1.5 ml centrifuge tube with 10 ul sterile deionized water.
2. Use a 10 ul petite tip to pick a quinoa sized yeast colony. My plate is about 2 weeks old.
3. Smear the colony inside the bottom of the 1.5 ml centrifuge tube and stir the water. You should see the water become murky.
4. Put the tube in -20 Celsius freezer for 2 mins.
5. Take the tube out of the freezer, vortex for 4 sec. Then put it in a 95-celsius water bath, boil 2 mins.
6. Take the tube out, vortex, then put in -20 Celsius freezer for 2 mins.
7. Take out, then put it in a 95 Celsius water bath, boil 2 mins.
8. Spin down the water inside from the top of the lid. Use a petite to make the liquid murky again.
9. Use 2 ul of the murky liquid in your 25 ul volume PCR reaction as the DNA template.
Hope this will work for you too!
I tried the microwave the cells for 1.5 mins protocol too. It worked but the band is not as strong as this protocol.
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