I am searching for options of templates for PCR reaction, to use the resultant PCR product as a template for in vitro transcription. I came through options such as plasmid containing gene of interest (GOI) and genomic DNA. Can I use first strand cDNA as a template for PCR amplification with primers (containing the T7 promoter sequence) specific to GOI ? Will this give a product incorporated with T7 promoter sequence which I can use for in vitro transcription?
yes you can use a PCR product with the T7 promoter sequence at it's 5'. how big is your fragment of interest?
@Siddharth Anand: The size is 1200bp. My question is that can i use first strand cDNA for the PCR to obtain a product with T7 promoter sequence incorporated in it?
Sure you can. I usually use the linear template kit from Qiagen. Moreover by using this kit you could add a His- or Strep-tag either on the C- or N-terminus or on both sides.
Best
How did that work out for you?
I do it all the time, it's far better than linealizing plasmid each time you have to make new probe. What i do is to clone in the pgemt easy vector using specific primers and then once i have that sequenced, i keep a good amount of plasmid to use as pcr template using primers for the sp6 and t7 promoters. That pcr product is the one I use as template for the probes. Make sure you get rid of all the DNA using DNAse before using the riboprobe.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods