Entering edit mode
6.1 years ago
farinaz
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0
hi. Dear all. I see not colored peaks in Sanger sequencing results after 300-450 bp. I checked sequencing reaction condition with different templates and specific primers, but didn't solve my problem. I have one band on gel agarose. Examples of this sequencing results attached with this post. In fact, after 300-450 bp, one gap is created and then the peaks dont have color. what is the problem?please help me that solve my problem.
Maybe the dyes bleached, did you protect your samples from light? No I am kidding, this must be something with your settings in the software, what software are you using? Can you try another software program? Like a free copy of Chromas?
Yeah this looks like some slightly weird rendering behaviour of that particular software (though I’m not familiar with it). You may need to speak to the software authors and file a bug report.
In the meantime, h.mon’s suggestion of some alternative software is probably best. SnapGene Viewer is also free and can read trace files.
Hello,
could you please post the Screenshots of the raw tab as well?
fin swimmer
screenshots of the raw data of three examples is posted for you. thank you of your help.!
Hello farinaz.cell88 ,
thanks for the screenshots. At first: You can recolor the peaks by going to the end of the sequence and do a right click into the area where the quality bars and the base letters are. In the menu that'll open choose
Set CR end ]for recolor until you mouse position.In your first example one can see a significant drop off of the signal. If this happens in "short" fragments, this is usually due to high GC content or sequence structures that can form a hairpin. Try to increase the denature temperature during the sequencing step.
In the second example the blue peaks looks a bit strange. They are higher than the other, which should not happen. Also in the screenshot the raw data looks a bit noisy. Does the raw data always look like this for this fragment? Have you done any clean up steps between PCR and Sanger Seq? Is there any additional weak band to see on the gel?
fin swimmer
hello finswimmer very thanks. your comments are very useful. l done "set CR end ]" and recolor my peaks. about purification after PCR, we purify templates with exosap enzyme and about clean up after sequencing reaction, we do ethanol percipetation method.