Posted 29 July 2009 - 04:56 PM
I am new to cloning, using Gateway technology.
I am using pGEM T as my donor vector with a 1.2kb insert. I have performed miniprep on the plasmid
Sequencing of the insert has the C-terminal truncated which means the primer at the end is missing, so therefore
it is useless since you need the two flanking att sites for cloning into the pENTR vectors for Gateway.
Does anyone know why the last 100-200 or so amino acids did not turn up when being sequenced?
Posted 06 August 2009 - 02:43 AM
If you have created a PCR product with the correct att sites attached you should perform the BP reaction directly into pENTR, no?
It has been a while since i did Gateway so I could be barking up the wrong tree here
Otherwise, to answer your question - could just be the sequencing ran out before then (or did you sequence from both ends, ie by using both M13F and R??) or it was dodgy so which ever program you were using to look at it cut it out (check the chromatograms). The ends of sequencing are usually crap.
You could easily check for the insert by doing a PCR on the plasmid prep using the primers originally used to create the insert in the first place
OR it could be that the E.coli cells you transformed into have recombinatory mechanisms that caused them to snip out part of your insert (not that likely compared to other things it could be)
OR it might just be one of those things that happen in the wonderful world of cloning
Edited by Arqwen, 06 August 2009 - 02:48 AM.