I was told to do PCR from the serial dilution of epiphytic and endophytic colonization sample that I did earlier. Question here is do I extract the dna first or just use the bacteria cultured into a broth directly for pcr?
Hope someone could help! Thanks in advance!
You can't use cell for PCR. You need to extract DNA. Do genomic or plasmid (whatever you need) DNA extraction. Methods are available online. Do not forget to quantify DNA using gel and nano-drop, sometimes nano-drop gives exaggerated readings. Then use purified DNA sample for PCR amplification. Also always keep at least one backup sample at every step, so if something goes wrong, you don't have to start over.
Once extracted the DNA you can amplify on different dilutions (1:10, 1:50, 1:100...) because genomic DNA could contain inhibitors of polymerase. Diluting your sample (you can do with water if you don't need to store your samples for long time), you reduce the negative effect of inhibitors without affecting (till a certain level) the amplification.
And actually you can use cells for PCR (but it's not clear to me your situation). Depending of the bacteria a 10 min step at 100°C is enough to extract DNA for amplification.
At the first you should extract your DNA from Dilutions.
I used this protocol since last year and it is so useful for 16sRNA to detect bacteria
very simple protocol : you should bring half of loop of your bacteria cultures and mix it in 1.0 ML distilled water then put it in boil water for 30 min after that centrifuged it for 30s in 3000. just this
But I used it just for 16sRNA with conventional PCR , not specific primers.
By the way you should use the good protocol.
good luck with your work
kaml sadeghi
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