is amplification directly from my ligation reaction possible? - (Oct/06/2006 )
The ligation reaction is made up of cDNAs and my vector. I am interested in obtaining more sequence information from only a single transcript and I have designed primers to do so once I have the transcript cloned. I am wondering if i can amplify directly from my ligation reaction. It would be so much simpler this way. I am worried that i wont have enough starting material though. any thoughts?
hmm.. my gut reaction to this suggestion is an instant "No"
Assuming only a single species of cDNA is in the ligation mix,
Any errors in the cDNA will be undetected. As it is the population of insert+vector molecules that's being sequenced rather then clones of an individual plasmid.
Also it feels odd, as the sequence data collected, won't be from the plasmid whcih is later obtained from the midipreped.