3 replies to this topic

[bandit]

Hello everybody!

I have got a problem. I did PCR and sequencing of nuclear Internal Transcribed Spacer (ITS) of a plant using two primers ITS1(forward) and ITS4 (reverse) . The PCR was quite successful. I employed the same primers for sequencing reaction and got two clean sequences (forward and reverse) without secondary peaks. I used the SEQUENCHER software to make the consensus sequence, however, the forward and reverse sequences appear in the same direction. Why the sequences obtained from forward and reverse primers didn't run in opposite directions in sequencher? PLEASE HELP. Thank you in advance!!!!!!

Sincerely,

[HomeBrew]

I'm not familiar with sequencher -- are you sure it didn't automatically reverse-complement one of the sequences to align them?

Do the original files (fasta and/or abi) from the sequencing facility agree that the sequences run in the same direction?

[elpollodiablo]

HomeBrew, on Nov 18 2009, 05:08 AM, said:

are you sure it didn't automatically reverse-complement one of the sequences to align them?

Sequencer does this when doing automatic assembly.

[bandit]

Thanks for your suggestion! Well, I get ABI files from the sequencing facility. I have had no problem in assembling forward and reverse sequences with SEQUENCHER when it's cpDNA. The problem occured only with the nrDNA.