wrong sequencing result?! - (Sep/15/2006 )
Hi,all!
I have newly transferred a 800bp fragement into a empty pGEX-5x-3. After the ligation, I have applied single digestion, double digestion,and everything is all right. So I took it to sequencing, BUT there are totally no matching with the gene I needed. No primer sequence. only one enzyme digestion site (EcoRI), the other enzyme digestion site (SalI) is missing. More wierd is that the fragement ligated into the vector is 100% matched with another gene on the website.
Hope I have said the problem clearly. I am really confused. I need your help!
Any suggestion is welcomed!
I have newly transferred a 800bp fragement into a empty pGEX-5x-3. After the ligation, I have applied single digestion, double digestion,and everything is all right. So I took it to sequencing, BUT there are totally no matching with the gene I needed. No primer sequence. only one enzyme digestion site (EcoRI), the other enzyme digestion site (SalI) is missing. More wierd is that the fragement ligated into the vector is 100% matched with another gene on the website.
Hope I have said the problem clearly. I am really confused. I need your help!
Any suggestion is welcomed!
Did you get the fragment from another plasmid? Has it been sequenced when it was in the other plasmid?
Did you get the fragment from another plasmid? Has it been sequenced when it was in the other plasmid?
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Yes, I think so. I got it from a lab member. She has ligated it to the pcmv vector. What I need to do is to PCR one part of it and transfer the needed fragment into another vector pGEX-5x-3.
So where is wrong? since the duble digestion is right which indicated that EcoRI and SalI both can digest it, but the sequencing result is totally different! I only got EcoRI and SalI is missing.
Perhaps your sequencing reaction has failed and you're getting cross capillary fluorescence from an adjacent capillary (the result from somebody elses sequencing).
Actually, the sequencing company failed for the first time, and had to try again to get this result!
However, our lab also got the plasmid containing the gene named "the sequencing result". So did there perhaps some contamination exist?
I would send the original plasmid for sequencing and I would sequence again the pGex containing the insert
I am going to send them for sequencing. Hope it will work out this time.
Does the sequence match a plasmid gene? This was a "liberate a fragment from one clone, and ligate it into another vector" experiment, right? Maybe you recovered the wrong fragment from the first digestion of the original clone...
Does the sequence match a plasmid gene? This was a "liberate a fragment from one clone, and ligate it into another vector" experiment, right? Maybe you recovered the wrong fragment from the first digestion of the original clone...
Do you mean that the original one contain two different plasmids? Does that mean the original one is contaminated by another plasmid?
What was the source of the 800 bp fragment that you cloned into pGEX-5x-3 and sequenced?
What I was suggesting was that if you got this fragment by digesting it out of another plasmid, you might have either recovered the wrong fragment or the 800 bp fragment you recovered was actually a mixed population of fragments, comprising your desired fragment and other ~800 bp fragments derived from the original vector.
In either event, the incorrect fragment you cloned would be a section of the original vector, and thus should be identifiable as such from its sequence.