troubleshooting + ligation

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Wed Nov 21 14:08:14 EST 2007


hi,
are u using any ligation kit?
whats the temperature of ligation and whats the time?
first thing to see is whether its ligated or not
only then you should proceed towards transformation.
try overnight ligation at 14 degrees and then..run an agarose gel to see
your product.
if its ligated it will become circular and its migration will be faster (u
can see supercoiled and stuff like that)
subject that again to single digestion..it will become linear and will match
with new mol weight (vector+1.5kb)
then try cloning it.

Sudheendra.

On Nov 21, 2007 10:51 AM, Anajli Thomas <anjalirthomas from gmail.com> wrote:

>      I have been trying to clone a 1.5 kb insert to pEGFP c1 vector. The
> restriction enzymes used were EcoR1 and Kpn1. I have tried ligation thrice
> and still no colonies. JM109 was used for transformation.
>
> The restriction enzymes were added together for cutting the insert from T
> vector (primary clone). For ligation I added 5 ul of insert and 2ul of
> backbone.
>
> Could you help me out with this problem
>
> Sincerely
> Anjali
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