10 replies to this topic

[Baroudeur]

Hi guys,

Anybody sees any drawbacks to performing PCR directly on a bacterial colony (ie. E. coli) ??

Edited by Baroudeur, 30 August 2011 - 02:05 AM.

[phage434]

It's called colony PCR, and very common. Major problems are false positives from ligation product passed through during plating, and false negatives due to inhibitors from the colonies. Since PCR is so sensitive, you can use remarkable dilutions of the colony to solve the inhibitor problem. Use a 10 ul tip to touch a colony, add to 50 ul of pure water and swirl. Plate a master plate with the same tip using 2 ul of the water and use 0.5 ul of the water (different tip) in a 10 ul pcr reaction.

[susanna]

Hi,

i was wondering the same thing:
1) so if you cloned a part of a gene in a plasmid, you can perform PCR straight on the plasmid, using the same forward and reverse primer you would use to amplify the gene on the patient samples?
2)Do you then need the plasmids to perform PCR, or is it possible to use the E.coli with the plasmid within as inputmaterial? Cause i assume that you first have to perform cell lysis of the E.coli bacteria to yield the plasmid?

greetz

[Adrian K]

Baroudeur, on 30 August 2011 - 02:04 AM, said:

Hi guys,

Anybody sees any drawbacks to performing PCR directly on a bacterial colony (ie. E. coli) ??

Agreed with phage434. For me, if I want to clone a gene from a bacteria, I will just use colony PCR instead of performing DNA extraction (although I do a quite different method from what phage434 suggested, not sure whether it is publishable...). The draw back would be reduced sensitivity, but it doesn't really bother's me in my case. IT all depends on your application.

susanna, on 31 August 2011 - 12:09 AM, said:

Hi,

i was wondering the same thing:
1) so if you cloned a part of a gene in a plasmid, you can perform PCR straight on the plasmid, using the same forward and reverse primer you would use to amplify the gene on the patient samples?
2)Do you then need the plasmids to perform PCR, or is it possible to use the E.coli with the plasmid within as inputmaterial? Cause i assume that you first have to perform cell lysis of the E.coli bacteria to yield the plasmid?

greetz

1) yes
2) if your cloned genes doesn't resemble any part of E. coli, you can possibly do that (I will suggest you run -ve ctrl by using just E.coli without plasmid). I usually use colony PCR to screen and select for my +ve clones. This is because in the first step of your PCR cycle where you are heating in 95C for few minutes, it is good enough to break down the bacterial cell wall.

Hope I answer your question

Rgs,
Adrian

[susanna]

oh well, thank you adrian for your answer.
How much bacteria do you then need as input for PCR reaction?

[susanna]

another question,
when i'll use the same primers (FP and RP) as i used for my linear patient DNA, on the bacteria, will i get linear DNA, although i'm amplifying from circular plasmids?
greetz

[Adrian K]

susanna, on 01 September 2011 - 01:06 AM, said:

oh well, thank you adrian for your answer.
How much bacteria do you then need as input for PCR reaction?

Not saturated enough to kill your PCR reaction. Usually 2-5ul of McFarland 1 density.

susanna, on 01 September 2011 - 01:09 AM, said:

another question,
when i'll use the same primers (FP and RP) as i used for my linear patient DNA, on the bacteria, will i get linear DNA, although i'm amplifying from circular plasmids?
greetz

The plasmid is circular because it joins together. In other words, if you can make your DNA fragment joints together you get a circular one, else, You will get linear DNA.

[Baroudeur]

susanna, on 01 September 2011 - 01:06 AM, said:

oh well, thank you adrian for your answer.
How much bacteria do you then need as input for PCR reaction?

Just take a pipette tip, carefully pick half a colony and mix it with your PCR rxn mix and you're ready to go ...

[susanna]

thanx

[afRNA]

I think below link will be helpful to you.
https://docs.google....vn6DLF64bpxxNtw

[susanna]

an

afaq.mohamed@yahoo.com, on 13 October 2011 - 07:21 AM, said:

I think below link will be helpful to you.
https://docs.google....vn6DLF64bpxxNtw

and if you use pure plasmids, extracted from their bacteria?