BioTechniques Molecular Biology Techniques Forums

by **Ayesha Kanwal** » Feb 28 2012 2:02 am

Why a fraction of an isolated colony of the suspect organism (bacteria) is transferred directly into the PCR mixture instead of template DNA? What would be better in case of Pasteurella multocida?

by **relaxin** » Feb 28 2012 9:36 am

Colony PCR is meant for quick screening without the need of purifying the DNA with miniprep. It also saves time in growing the bacteria.

by **CrowSan** » Mar 02 2012 7:28 am

And of course by transfering only a fraction of the colony to PCR the other part of the colony can be transfered to media for storage. If the PCR is positive the rest of this colony can be grown up.

by **GelBandIt** » Mar 03 2012 10:45 pm

IME, only an invisible amount of cells is necessary for colony PCR. If you touch the colony with a pipette tip then swipe the tip around inside the PCR tube before adding the master mix, you will probably have enough template.

Some protocols involve suspending a colony in ddH2O then boiling to lyse the cells before diluting the mixture for use as a template. I've never done that.

by **Aarfy** » Apr 11 2012 3:47 pm

GelBandIt wrote:Some protocols involve suspending a colony in ddH2O then boiling to lyse the cells before diluting the mixture for use as a template. I've never done that.

We typically sample using a pipette tip and place it directly into the PCR Master Mix and increase the time of the initial denaturing step to ensure lysis of the cells.

by **nestaB** » May 22 2012 5:59 pm

Are you actually pippetting the colony sample? If so I suggest you use a low-binging or low-retention pipette tip.

by **way2jordan** » Aug 17 2012 2:27 am

If the cells are oldish (growing for >16hrs) I would avoid using them for PCR. This is true for both colonies and cultures.. Cells grow more than 16hrs greatly increase their production of liposaccarides (slime!) which interfereswith the PCR reaction. Otherwise (provide sufficient dilution) and the cells are 'fresh-young' there is no difference between the two methods. Personally I prefer doing my PCR on the colonies.. I use colony PCR for screening purposes and doing the screen on colonies saves me a day.
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pipette :roll:

BioTechniques Molecular Biology Techniques Forums

Transferring isolated colony directly into the PCR mixture.

Transferring isolated colony directly into the PCR mixture.

Re: Transferring isolated colony directly into the PCR mixtu

Re: Transferring isolated colony directly into the PCR mixtu

Re: Transferring isolated colony directly into the PCR mixtu

Re: Transferring isolated colony directly into the PCR mixtu

Re: Transferring isolated colony directly into the PCR mixtu

Re: Transferring isolated colony directly into the PCR mixtu

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