troubleshooting + ligation

[Jose de las Heras]

"Anajli Thomas" <anjalirthomas from gmail.com> wrote in message 
news:mailman.696.1195669227.23109.methods from net.bio.net...
>      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

If somebody asked you this, do you think you have all the necessary 
information to be able to suggest anything helpful?

You say nothing of how you purified the fragments, how teh digests looked... 
but what really gets to me is the "For ligation I added 5 ul of insert and 
2ul of backbone". Even if I know the size of pEGFP, talking microlitres is 
pointless as I don't know teh concentration.

It's one pet hate of mine, when people write protocols etc talking of 
"microlitres" as if we all knew magically what concentration of reagent 
they're using. It makes me think that they don't know. It makes me think 
that they don't really know what is important.

Sorry to sound a bit harsh... but I feel it's got to be said.

Just think about the problem a little bit before you post, so that you're 
able to give the relevant information, and I am sure you will receive more 
helpful answers.

Regards,

Jose de las Heras