Question about reverse transcription of bacteria mRNA - (Oct/15/2005 )
Hi,
I am new in molecular biology. Could someone tell me how to reverse transcript mRNA of bacteria to cDNA. Thank you
-Wenxi-
First, you isolate the RNA from your bacterial culture (there's a lot of commercial kits available to do so). After this, you set up a reverse transcription reaction (once again, a lot of enzymes available from different companies), using primers that you design or choose. You can choose between oligodT (with or without an extra A, G or C) primers, random primers or gene specific primers depending on your needs. The RT reaction mostly takes an hour or less.
You better also perform a negative control reaction to make sure you have no contaminations.
-vairus-
QUOTE (vairus @ Oct 16 2005, 01:08 AM)
First, you isolate the RNA from your bacterial culture (there's a lot of commercial kits available to do so). After this, you set up a reverse transcription reaction (once again, a lot of enzymes available from different companies), using primers that you design or choose. You can choose between oligodT (with or without an extra A, G or C) primers, random primers or gene specific primers depending on your needs. The RT reaction mostly takes an hour or less.
You better also perform a negative control reaction to make sure you have no contaminations.
You better also perform a negative control reaction to make sure you have no contaminations.
See that it's reverse transcription of mRNA from bacterial, we must know that procaryotic mRNA lacks poly (A) site, so oligo dT is not suitable for such purpose, random primer maybe more suitable. By the way, because there is no intron in bacterial genomic DNA, why not use genomic DNA directly?
-zhongmindai-
QUOTE (zhongmindai @ Oct 16 2005, 06:39 AM)
QUOTE (vairus @ Oct 16 2005, 01:08 AM)
First, you isolate the RNA from your bacterial culture (there's a lot of commercial kits available to do so). After this, you set up a reverse transcription reaction (once again, a lot of enzymes available from different companies), using primers that you design or choose. You can choose between oligodT (with or without an extra A, G or C) primers, random primers or gene specific primers depending on your needs. The RT reaction mostly takes an hour or less.
You better also perform a negative control reaction to make sure you have no contaminations.
See that it's reverse transcription of mRNA from bacterial, we must know that procaryotic mRNA lacks poly (A) site, so oligo dT is not suitable for such purpose, random primer maybe more suitable. By the way, because there is no intron in bacterial genomic DNA, why not use genomic DNA directly?
Thanks a lot. I want to look for the genes responsible for some chemicla treatment with bacteria.Could you tell me how to do experiment with genomic DNA directly? Thanks again.
Wenxi
-Wenxi-