I have amplified 1.04kb and 1.16 kb fragments, then I went for touch down PCR by using these two fragments as template to amplify the 2.2 kb of my desired gene. I got amplification of 2.2 kb with less intensity and a one of the fragments was more intense and is amplified (1 kb) , so I diluted the PCR product 1/10 and kept normal PCR with annealing of 40- 58 C. It is also showing the same kind of amplification (I used pfu poymerase). Please suggest how I can overcome this.
Why are you trying to amplify 2.2kb from two separate fragments? And is your result really so surprising if you consider what is happening in the PCR? Why not just run a single PCR for the 2.2kb gene? You can still use a nested system if needs be to get amplification. You could even approach this opposite as you have done (as it seems you have the primers): amplify the 2.2kb then two nested PCRs for the 1.04 and 1.16kb fragments, sequence and join to get 2.2kb!
Hi, first of all I agree with Russell Scott, try to amplify 2.2 kb direct, but most likely it appears that you want to concatenate/join two different genes amplified from different locus. Are your PCR products' ends are compatible for joining? I was successfully able to concatenate 5 different exon even from multiplex PCR by using compatible end describe in by Tuohy and Groden (1998), the same article is attached. Hope this may help you, Best luck.
Hello guys,
I tried to do 2.2 kb amplification when i couldnt get, then i moved into amplify the two fragments and trying to join them coz those ends are compatible by 4 aminoacids.
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Annealing