Problem with colony PCR - (Oct/16/2006 )

hai friends,

i am veeresh from austria working on Pichia i have problem with colony PCR it works some times fine but it is not working well with me can any body find solution to my problem i am using Phusion polymerase for the reaction

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Why on earth are you using Phusion for a colony PCR? Isn't it a bit too expensive compared to regular Taq?

Please give us details on your procedure, how much e. coli are you adding to your final PCR, what controls (both positive and negative) do you take along with you and are they behaving like they should (in the case your colony-PCR doesn't work). Have you checked if the plasmid was there and correct (by restriction digest or sequencing) in case your colony PCR was negative etc...

With the information you've given us, we can't help you...

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To add onto this matter :-
Please don't use Phusion for colony PCR. Aside from the cost, Taq has far better yields for short PCR product then proof reading enzymes. Taq doesn't waste time with 3'nuclease activity.

Now as vairus has commented, please give us more details.
I would like to know
-what strain of e coli are you using
-how old (in hours) are you colonies when you pick them for colony PCR
-what method of picking do you do... colony from plate or colony from growth medium
-If picking from plate, what do you use to pick the colony
-how much cells do you add to the PCR reaction mix
-How large if you PCR reaction mix
-what is your PCR mix composed off
-What are your cycling conditions
-how many cycles do you do?

Basically you have to tell us exactly what you did, and please describe how each stage looks like.

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believe it or not, i at last discovered what was the problem with my "sometimes working and sometimes not" PCR. it was different wells on the cycler. my PI couldn;t believe it and he doesnt till now . i put in raw 1, nothing, put in raw 2 bands are there....

but very important is just to touch the colony, put this in 50ul distilled water, and take only 1ul of that mixture for PCR. I also heat the mixture to 100C for 10 min. some say this step is not necessary.

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Kathy, i believe you. There are two wells on my thermal cycler that don't work. There must be some problem with the heating block as any PCR mix placed in the two well will be evaporated no matter the precautions. Time for servicing?

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Hi,

If you're doing colony PCR with Pichia, and the PCR is inconsistent, could probably be the lysis that's giving you the problem. Haha... your Taq is definitely very expensive. Anyway, I've got this manual from Invitrogen in the net that explained how to screen Pichia with PCR:
http://www.invitrogen.com/content/sfs/manu...yselect_man.pdf.

But you have to also ensure that the primers you used are OK. Can try PCR with genomic DNA or DNA (higher purity) and see if the bands are "thick". Assuming that PCR conditions are optimised, especially the initial denaturation time for genomic DNA.

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you mean the mix actually evaporates??? no i think this is not my case. no evaporation here. never checked the loss in volume though. it's just nothing on the gel...

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Yup, it evaporates. Nothing behind. Gone. Something must be wrong with the temperature control. I am afraid the whole row of wells maybe affected but I am not willing to do the experiment to find out, I just avoid that row for my experiments.

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(1) I think Pichia do not as easily lyse as E coli, would 100oC 10min is eoungh for giving enough genomic DNA from the cell (gene were insert into the genome of Pichia in your case)?

(2) Is genomic DNA less easily amplified ?

(3) What is the medium of your agar plate and the selecting antibiotic ? Some of these would inhibit DNA polymerase (the medium for yeast is relatively high salt ?)

(4) I don't know

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