2 replies to this topic

[Retinoblastoma]

Hi, I have been doing sequencing of 18srRNA obtained by using specific primer from PCR from algae. My PCR product seems good in agarose gel. After sequencing the initial nucleotides can be observed distinctly but as we go further reading the sequence smear appears all the time. I optimised the concentration of the gel, used new primers, and even the Dynabeads, the result is same. How can i get rid of this smearing? What other factors can affect the sequencing? Its my fifth attempt to get the correct sequence but cant get rid from the smears.
It would be grateful if someone could help me out to come through these problems.

[Micro]

Hi, you seem to be using the term "smear" in reference to a gel and to a sequence reaction. (1) Are you doing an old style gel based sequencing run not the more recent capillary method? Or (2)are you observing smears in your gel that you then sumit for sequencing and are getting poor sequencing reads?

If (1) not sure about how to fix this but if (2) you might want to try changing your thrermocycling parametres (i.e. increase annealing temperature or doing a touchdown PCR) that is if it is the first PCR that is causing the problem (which it might not be). Otherwise there is another thread discussing called "Cycle sequencing (Big Dye)" that began recently, that might be helpful if the probelm is being caused by your sequencing reaction causing poor reads.

Cheers
M  

[mdfenko]

if the smear is what i think it is, then some sequencing kits add some 7-deaza-dgtp or ditp (an analog of dgtp) to reduce "compressions".

see these attachments

Attached Files

•  7-deaza-dgtp.pdf   53.97K   305 downloads
•  ditp or 7-deaza-dgtp for sequencing.pdf   282.43K   206 downloads