Sequencing incorrect after digestion and ligation - (Jun/30/2008 )
I was wondering if anyone can help me. I am at my wits end. I am working with a cassette comprised of a promoter, g10 leader sequence, my gene of interest and a terminator with a pcr2.1 backbone. I digested out the entire cassette with sites outside the promoter and terminator and ligated it into pcr 4 Topo (using Klenow). I checked it by digest to make sure that the cassette had ligated into pcr 4 topo and that the sites I am working with were still present and it gave me bands of the correct size.
The cassette was made by a person who was working in the lab before I arrived so then I just digested out their gene of interest and replaced it with my own. I also checked this by digest and it gave bands of the correct size as well, indicating that everything was fine.
I sent it for sequencing just to be sure (to 2 different companies). However, the sequencing results are all over the place. My gene of interest is intact and correct but the promoter and the terminator are full of mistakes, base changes, deletions and insertions. The cassette was sequenced before and was correct so I don't understand what has happened.
I would really appreciate any help that anyone can give me. I have asked a lot of people in my lab and the lab next door but no one can help me.
The cassette was made by a person who was working in the lab before I arrived so then I just digested out their gene of interest and replaced it with my own. I also checked this by digest and it gave bands of the correct size as well, indicating that everything was fine.
I sent it for sequencing just to be sure (to 2 different companies). However, the sequencing results are all over the place. My gene of interest is intact and correct but the promoter and the terminator are full of mistakes, base changes, deletions and insertions. The cassette was sequenced before and was correct so I don't understand what has happened.
I would really appreciate any help that anyone can give me. I have asked a lot of people in my lab and the lab next door but no one can help me.
Did you start with a cryopreserved culture or the DNA of your predecessor? Can you go back to the original cassette that was used by your predecessor? I would hope they at least have the empty cassette (or whatever they started with) transformed in E. coli. Depending on how they maintained their strain the mutations you see in the cassette could have accumulated over time. Many years of serial subculturing overgrown cultures could be one explanation.
When was the cassette sequenced? If you haven't already done so, sequence the cassette from the original vector. This will tell you if the cassette was already mutated before you got it or if it has become mutated during your cloning process. Alternatively, you mentioned there are deletions and insertions in the sequence. You can use this information to carefully design a restriction screen that will tell you if the mutations are in the original cassette or just your intermediates and the final clone (use the final clone as a positive control). Choose restriction sites that will be missing in the sequence if it is deleted.
There is more to do based on what happens next. Keep us posted and i'll let you know what to do next based on the results from the above experiments (should you choose to do them).
I grew up the original cassette from a glycerol stock. I also sent it for sequencing with my own cassette to make sure that there were no mistakes in it to start with and the sequence is perfect.
The cassette was made by a person who was working in the lab before I arrived so then I just digested out their gene of interest and replaced it with my own. I also checked this by digest and it gave bands of the correct size as well, indicating that everything was fine.
I sent it for sequencing just to be sure (to 2 different companies). However, the sequencing results are all over the place. My gene of interest is intact and correct but the promoter and the terminator are full of mistakes, base changes, deletions and insertions. The cassette was sequenced before and was correct so I don't understand what has happened.
I would really appreciate any help that anyone can give me. I have asked a lot of people in my lab and the lab next door but no one can help me.
Just to be certain, when you're looking at your sequence, are you looking at the end of the read? The ends of the read - close to the primer or near the end of what was sequenced - will often contain lots of errors.
No, I sequenced with both a forward and reverse primer, so I am ignoring the ends of both reads.