full length gene isolation.

[prabuddhasarkar]

Hi everybody
I am back after a long break. A good news is I have completed sequencing the whole gene target of mine. But that was done in 3 parts. Now I have designed primer from the 3' and 5' UTR, to amplify the full length geneas a single fragment. But after 2-3 nested PCR with deep vent, I am getting a strong band just next to the well of the gel folloed by high to low mol wt smear. But my target is of 1.8 kb size. Please suggest, if you are getting these kinds of results and what to do next. and I have used a gradient temp ranging feom 48 C to 61 C in case of all the PCR.

Thanks in advance
Prabuddha Sarkar
Research Scholar
Visva-Bharati
Santiniketan
India

[prabuddhasarkar]

Sorry, the target size is 2.8 kb.

[pigfarmer]

Exactly how long did it take you to sequence 2.8kbp?

[prabuddhasarkar]

See, I don't have a sequencer in my dept. And there are fund limitations, so it took almost 3 years to get the full length gene sequenced. I had to send the samples to other places. Anyways, did you encounter problems like mine which I have asked, if yes, then please suggest me.

[Warthaug]

Are you amplifying from a plasmid, genomic DNA, or other?

The bright band next to the well is most likely your template - i.e. genomic DNA or a plasmid.

Smears are usually due to excessive amounts of template; low-affinity interactions between primers and template occur under these conditions, leading to all sorts of amplifications occurring. Reducing the concentration of template should help with that.

Another possibility may be template degradation (if your template is old, or not stored properly). You may need to make a new stock of template in this case.

Lastly, you may have some contaminating DNA. This usually comes from the water you use in the PCR reaction. Make sure you're using molecular-grade water.

Hope this helps

Bryan

[prabuddhasarkar]

Thank you Bryan for your reply. Actually I am using total cDNA as template which was synthesized by oligodT primer from total RNA. And the primers I am using was designed from the 3' and 5' UTR region of the sequenced gene. The templates for the later nested PCR are the diluted PCR products from the previous round of PCR. Regarding the template concentration, I have used 500 times diluted template. but the same thing happened. But the water contamination you are referring could occur, I will check that. Any other suggestions?