advice on DNA extraxtion: boiling method - (Aug/22/2007 )
hello,
i am trying to identify marine bacteria from sponge and i am using 16S rRNA gene analysis.
my problem is that there are 2 method used to extract the DNA
1) direct colony PCR
2) boiling method
the result that i get was not very satisfied due to smearing and more than 1 band.
can someone help me or give me some advice.....PLEASEEEEEEEE........ 
i am trying to identify marine bacteria from sponge and i am using 16S rRNA gene analysis.
my problem is that there are 2 method used to extract the DNA
1) direct colony PCR
2) boiling method
the result that i get was not very satisfied due to smearing and more than 1 band.
can someone help me or give me some advice.....PLEASEEEEEEEE........
What do you want: RNA or DNA
When you want to check rRNA you have to make RNA isolation and check by gelelectrophoresis
When you want to check DNA content: you can make colony PCR or boiling method. i think in boiling buffer is RNAse?
The problem is likely the PCR cycling conditions rather than the DNA sample. You could also be adding too much template, a common problem. Less is more. Try reducing the template amount and raising the annealing temperature (or a gradient PCR if you can do it).
thanks for the advice. i will try and report back after i continue my lab work here. thanks. if there any other suggestion, i will surely appreciate it.
because you boil your colony, so there might be some other 'junk' in the genomic you extracted. when you do PCR, these "junks"might have effect on the PCR.
i used to do the exactly same thing as you. boil colony and PCR 16s rRNA. same thing happen. my primers are very specific but i couldn't get any band. after that i add PCR additive in my PCR mixture (mixture contains 10% glycerol, 10% DMSO, 10% formamide)... add 25% of PCR additive mixture into PCR reaction. then, my PCR has a very good yield.
works well for me everytime i PCR. it never fails me.
i used to do the exactly same thing as you. boil colony and PCR 16s rRNA. same thing happen. my primers are very specific but i couldn't get any band. after that i add PCR additive in my PCR mixture (mixture contains 10% glycerol, 10% DMSO, 10% formamide)... add 25% of PCR additive mixture into PCR reaction. then, my PCR has a very good yield.
works well for me everytime i PCR. it never fails me.
now thats interesting. can u tell me a bit more about it. how the PCR additive's component function during the process. and thus the pcr additive can be used the same way if i'm going to do colony-pcr. how about if we put proteinase K?