Reverse Sequences - (Sep/24/2009 )
Hi all
a questions regarding the orientation of sequences during cloning - a few months ago i cloned some PCR fragments (Topo10, thermal method), and the sequences came back as expected (aligned perfectly with the reference sequences).
A few weeks ago a new set of clones (different PCR product, Topo10, electroporation method) were sequenced, and nearly half of the sequences needed to be reversed for alignment (after which they aligned perfectly).
What causes selection of strand orientation during the cloning process? Should i try to improve PCR eficiency, ligation or what?
Has anyone else had similar experiences?
-Mulletman-
Mulletman on Sep 24 2009, 11:36 AM said:
Hi all
a questions regarding the orientation of sequences during cloning - a few months ago i cloned some PCR fragments (Topo10, thermal method), and the sequences came back as expected (aligned perfectly with the reference sequences).
A few weeks ago a new set of clones (different PCR product, Topo10, electroporation method) were sequenced, and nearly half of the sequences needed to be reversed for alignment (after which they aligned perfectly).
What causes selection of strand orientation during the cloning process? Should i try to improve PCR eficiency, ligation or what?
Has anyone else had similar experiences?
a questions regarding the orientation of sequences during cloning - a few months ago i cloned some PCR fragments (Topo10, thermal method), and the sequences came back as expected (aligned perfectly with the reference sequences).
A few weeks ago a new set of clones (different PCR product, Topo10, electroporation method) were sequenced, and nearly half of the sequences needed to be reversed for alignment (after which they aligned perfectly).
What causes selection of strand orientation during the cloning process? Should i try to improve PCR eficiency, ligation or what?
Has anyone else had similar experiences?
When you clone a PCR fragment the orientation in the clone is random. Just look for your primers in the sequence. If your forward primer is still in the same orientation than you don't need to complent inverse your sequence.
Regards,
Jackson
-Jackson-
Yes, direction of insertion into TOPO is completely random, no RE sites used....
-Chimp-