Colony PCR with Phusion - (Jul/31/2008 )
Hi everyone, I will appreciate any help.
I am sequencing a DNA fragment. In the beginning I was trying to sequence the PCR fragment directly from Phusion PCR. To do this I was using allele-specific primers. However, it turned out that the allele-specific primers were not that specific at all. Often I would get double peaks in the pherogram through out the whole fragment (this because I usually used an indel to create primers with different sequence that would distinguish between the alleles). Anyway, I soon changed to cloning. It worked just perfect, but turns out now my project is running low on funding and we cannot afford the plasmid purification kit any more. I tried the "old school" method, but I lost the DNA pellet more often than not, not to mention it took me half a day to perform the protocol. So then I decided to sequence the product of my colony PCR. So first I would screen my colonies through Taq colony PCR and then perform a second colony PCR with Phusion on the colonies that showed the right band on the first colony pcr. At the beginning it worked like a charm, but soon quality dropped really bad. So I tried to check the quality of my Phusion colony pcr through agarose gel electrophoresis. It turns out I had no band whatsoever. So I performed a gradient colony pcr (and here is where it gets spooky), found really nice bands around the temperature I was already using and decided to increase the temperature a couple of degrees to get better yield (?) because Phusion usually uses higher temperatures than Taq and I was using the same temperature for both. When I ran my colony pcr with all my samples, I got nothing again. I performed the gradient pcr three times, and I always get the same temperature range with really nice bands, but when I repeat the colony under the same conditions, I get empty lanes every time. It's really driving me crazy, all conditions are the same except it takes me considerably longer to perform the colony pcr with all my samples (the step where I have to pick each colony and put the toothpick in the master mix). And it cannot be the thermocycler because everyone is using it and they get bands.
Any help is welcome.
Failure of colony PCR, typically is caused by using too much cells in the PCR reaction. I would recommend picking the colony and diluting it with sterile distilled water (50ul) and use about 2.5ul for the PCR reaction. A multichannel pipette and a 96 well plate will be helpful.
Also note, since you are only sequencing plasmids, you can sequence using Taq PCR product. While the Taq will make mistakes, the mistakes will be distributed all over the sequence. Howevever when you look at the sequence read, you are looking at the average sequence. And this average sequence will refect the real sequence. So no need for Phusion.
Lastly, alkaline lysis miniprep plasmid extraction, after a while you will get good at it. It just takes a bit of practise.
Note: Phusion is not as active as Taq for short PCR products. So for PCR products (less than 1kb) Taq will yeild more PCR product than phusion. If you have money still on you, try using KOD hifi instead of Phusion, you get more PCR product (if the PCR product is less than 5kb, KOD hifi is more efficient).
Thanks for the tips. I will try this "bacteria dilution" thingy and see how it works. At the moment I'm starting to sequence Taq product. I think the same as you, that the mistakes of the Taq shouldn't be a problem, but people at my lab are a bit "fundamentalist" on just sequencing Phusion amplicons.... Anyway, I don't care, I just want to finish the sequencing asap.
So thanks a lot!
i don't know if it helps but here is an observation that i recently made with colony PCR:
usually, when performing PCR i have a 94°C denaturation step with the duration of 10s , which was always enough (also in GC-rich templates), saves some time and also the polymerase is more active because it gets less heat-killed. however, when i assigned this protocol change also to colony pcr I suddenly observed only ~1/6 of positive clones as before. when I changed back to 94°C/30s all was perfect again (with clones from the same plate!). So i think, a sufficient time of denaturation plays an important role in colony PCR. Maybe there is some cellular dirt in the reaction which sticks to the dna and inhibits primer binding which is only efficiently removed if the denaturation is ~30s.
interesting.
My colony PCR has a preheat of 95 Celsius for 4mins. And 94 Celsius for 30 sec for melting.
My colony PCR has a preheat of 95 Celsius for 4mins. And 94 Celsius for 30 sec for melting.
Mine also works like this. I will try the dilution step today, stay tuned for results....
If you read the Phusion manual, you will discover that the denaturing temperature recommended is 98C. Follow the directions -- the high temperature is important for Phusion to work, and will also be better for breaking open the cells.
I had already tried that one, no good results. I have to tell that I tried diluting the bacteria before adding them to the pcr master mix and it worked like a charmed I get nice bands in all the colonies that I had previously screened as positive.