sequencing problem - (Jan/10/2008 )
Recently I did a cloning and restriction digestion analysis showed my insert is there. But I did sequencing 4 times and all the time I did not get a good sequencing result. It reads only 100-120 base pairs and the sequence is almost 90% similar to original sequence of insert and rest 10% are additional base pairs or missing base pairs or unreadable (N) base pairs. It was a PCR cloning and I used pfu for amplyfying my insert. Any one of you please guide me where could be the problem?? Thank you
if sequencing is not good, then how can you rely on its results and think there are additional bases or bases missing. Try to get the sequencing right and then if you notice differences, there is some error for sure.
I meant on the initial part of my insert which is red during sequencing (in 100-120 base pairs) they are OK with 10% of error. I wonder if it is because I am not preparing my construct for sequencing, enough pure? The size of my whole insert is 800base pairs. Sorry for not explaining it properly.
Hi!
I used to have the same problem. How are you preparing the DNA, columns? And did you try a sequencing primer in the middle of the construct and at the 3´end? That helped me a lot. Depending on the sequencer, you need very good DNA, Mini Prep DNA for example doesn´t work for me.
Hope that helps
Yvonne
If the raw data is not clean or doesn't have a good signal, I would still consider it sequencing error than anything else. Check with the sequencing center, if the signal is good that it is interpretable? they could provide with additional information on the problem.
You need clean DNA for sequencing, if not you could get weird signals.
[quote name='Smarty1981' date='Jan 10 2008, 10:43 AM' post='122294']
Hi!
I used to have the same problem. How are you preparing the DNA, columns? And did you try a sequencing primer in the middle of the construct and at the 3´end? That helped me a lot. Depending on the sequencer, you need very good DNA, Mini Prep DNA for example doesn´t work for me.
Hope that helps
Yvonne
[/quot
Thank you very much for your reply. I am isolating the DNA by column and I am using forward and reverse primer just before the beginning and end of the construct. I just wanted to check the ligated region as well. What about first phenol.Chloroform and then column purification?
I am also concerned about purity of my DNA. What about Phenol. Choloroform and then Colum purification? This is what suggested by sequencing people. Thank you
You need clean DNA for sequencing, if not you could get weird signals.
I purify through columns and have the wash buffer on the column for 5 min. before centrifuging it. This has worked for me. If the sequencing people specifically ask something, stick to it. They know it better.