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Colony PCR

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Colony PCR

Postby Nmr » Sat Mar 19, 2011 7:22 am

Hi Dear friends

First, i would give you an idea about my question about... i'm going to identify and characterize several bacterial isolate from soil via 16s rRNA.
My question is. Is it possible to use the colony PCR in this case?... that mean i'm not going to extract the DNA from the colonies cell. as i understand from the paper that i read before which they used the bacteria grown in broth media in PCR reaction immediately without extraction the DAN.

so, what do you think is it possible in my case?..because i'm new in this type of PCR, i have never used before OR should i follow the tradition procedure of PCR.
plz i want your recommendation and suggestions as soon as possible.

Thanx
Nmr
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Postby JackBean » Sun Mar 20, 2011 10:58 am

I think it should be possible. However, the RNA is rather unstable and thus boiling the cells migth disrupt it, for that reason, you should probably first try with some pure isolate, which you know, it should work for 100%
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Postby canalon » Mon Mar 21, 2011 11:12 pm

Yes it is possibly to get a PCR from colonies or broth without purification. It is not entirely reliable, but 16S primers are quite robust, so that should be OK. I would use very little of the colony or broth as they can inhibit the reaction (1µl in 25µl isgenerally OK)

If you amplify from a colony you will have one species, and it can be sent directly for sequencing, but remember most of the soil bacteria will not grow in broth or on plates. If you use broth directly it is even worth since you will bias your population, amplify a mix of species that will need to be separated by something like DGGE or cloning.

And Jack when talking about 16S amplification/sequencing in bacteria, the implication is that the gene(s) for that specific RNA is amplified, not the RNA.
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Postby Nmr » Tue Mar 22, 2011 5:18 pm

Hi friends

first, thank you for the replies... second, from i learn about the colony PCR the process is quite similar to the normal PCR and even the processes are faster. and i have discussed with my friend who had an experience in molecular biology just as you guys, he told me that i will use a particular kit for colony PCR in order to amplify the 16s rRNA i should just follow the protocol.

and for the broth i will use an certain media consist aliphatic compound that the bacteria degrade the compound in order to grow. in this case it could be several genus of bacteria i have to identify.
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