I have recently cloned a gene into a SpeI/XhoI restriction site in a pME6032 vector and when I check it by sequencing and comparing it with the reference sequence. I noticed that I read my sequence in a 3' to 5' direction using a forward primer (even without making it into its complementary strand).
I can also check my insert by colony PCR and double digestion so I know that my insert was successfully cloned.
Anyway, I also noticed that my tac promoter region is oriented in a reversed direction when it was annotated in snapgene. I hope you can provide some insights to this.
Thanks!
Kris, according to my search there's no SpeI site in the the pME6032 vector. ¿Is that possible?
Estanis
Usually in directional cloning cant be cloned in reverse orientation unless the ends of two different restriction enzymes are compatible to show enough complementarity to get it cloned. following may be the reasons you cross check it
1. ends of restricted sequences are compatible
2. it can confirm the orientation of your cloned gene by PCR and restriction digestion
3. since you confirmed your gene integration by restriction it might have restored bothe the recognition seq of restriction enzymes.
4. if have promoter in reverse orientation as like you gene it possible that your gene may show expression provided that it start with AUG of your gene and not from vector
4. Another complication is protein induction and the host your using for protein expression.
Dear Kris
I think at first you should check the direction of your gene by PCR-Sanger seq with the new primers that located before or after of joint ends of your insert (on your vector).
but changing the direction of insert with different restriction sites in cloning procedure is not possible (if these enzymes do not produce the complementary ends) .
Amir
I think the collaborator introduced the speI site by pcr and cloning. my gene of interest was an exotoxin tagged with a 3FLAG sequence in this manner. SpeI-3FLAG-XhoI-Exotoxin-EcoRI. I tried to replace the 3FLAG sequence with a gene encoding a fluorescent protein.
anyway, thanks for all your answers.
I just mapped the plasmid and sequenced using another primer. I just figured out that the tac promoter and the lac operator for this plasmid runs in a reverse order compared to the ori site, so that might explain why this is so. anyway, I think the direction of transcription is like this
tac promoter -> lac operator -> EcoRI -> Exotoxin -> SpeI -> 3FLAG -> XhoI -> pOri site.
Its just that I can only read a portion of the gene sequence at a time so I have to use multiple sets of primers.
Anyway, thanks for all the help!
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