3 replies to this topic

[yhafezi]

Hello,
I am trying to do a very standard subcloning and getting results such as:
after ligating, transforming, and getting colonies, the mini-preps show no insert in the diagnostic cut (even though the ratio of colonies compared to the background plate looks great)
But when I sequence these mini's the insert is there! It can't be the enzymes because, I have replaced them. Also, they put the insert there in the first place.
Has anyone had problems like this before?? Any ideas??
Thank you!!
yassi

Edited by yhafezi, 06 October 2004 - 12:09 PM.

[SVTX]

Hi,

Try not to use the same enzymes that you had used to ligate the insert when testing for positives.  Sometimes, when the insert ligates to the vector, the sites get modified and may no longer be able to cut with the original restriction enzymes.  Hence, choose an enzyme/enzymes outside of your cloning sites.

SV

[solarisc]

I am sorry i am a newbiew..but I am curious what is the purpose of subcloning?  Why couldn't you just clone it straight into the vector you wanted???

Thanks for answering

[yhafezi]

Hi, I am also a newbie but from what I understand, after a molecule has already been discovered and cloned the first time, if you want to make a new construct (for example to put a different tag on it), you call it subcloning. Please, someone correct me if i'm wrong!
In response to the other reply tho, the sequencing shows the restriction enzyme sites to be good.
yassi

Edited by yhafezi, 07 October 2004 - 03:07 PM.