Sequence direction same - (Nov/15/2009 )
Hello!
I have got a problem. I did sequencing of Internal Transcribed Spacer (ITS) using two primers ITS1 and ITS4. I used the same forward and reverse primers for sequencing reaction. I anticipated to obtain two sequnces (forward and reverse). But when I used the sequncher software to make the consensus sequence, the two sequences have the same polarity. I retried but always got the same result. Basically the sequences obtained from forward and reverse primers should be in opposite direction. I can not understand why it always happened with ITS region. I have no problem with cpDNA sequencing and get the sequences in opposite direction with cpDNA sequencing. PLEASE HELP. Thank you in advance!!!!!!
-aashraya-
aashraya on Nov 15 2009, 06:43 PM said:
Hello!
I have got a problem. I did sequencing of Internal Transcribed Spacer (ITS) using two primers ITS1 and ITS4. I used the same forward and reverse primers for sequencing reaction. I anticipated to obtain two sequnces (forward and reverse). But when I used the sequncher software to make the consensus sequence, the two sequences have the same polarity. I retried but always got the same result. Basically the sequences obtained from forward and reverse primers should be in opposite direction. I can not understand why it always happened with ITS region. I have no problem with cpDNA sequencing and get the sequences in opposite direction with cpDNA sequencing. PLEASE HELP. Thank you in advance!!!!!!
I have got a problem. I did sequencing of Internal Transcribed Spacer (ITS) using two primers ITS1 and ITS4. I used the same forward and reverse primers for sequencing reaction. I anticipated to obtain two sequnces (forward and reverse). But when I used the sequncher software to make the consensus sequence, the two sequences have the same polarity. I retried but always got the same result. Basically the sequences obtained from forward and reverse primers should be in opposite direction. I can not understand why it always happened with ITS region. I have no problem with cpDNA sequencing and get the sequences in opposite direction with cpDNA sequencing. PLEASE HELP. Thank you in advance!!!!!!
You mean consensus or contiq? as far as I concern, you should do a contiq and then get the consensus... correct me if i'm wrong.
-adrian kohsf-