Need advice

J. Arturo García-Horsman arturo.garcia-horsman at uku.fi
Tue Apr 10 01:09:58 EST 2001


"Dr. Hiranya S. Roychowdhury" wrote:

> I am trying to get two ~50bp inserts, each with a sticky and a blunt end to
> ligate to a vector (sticky). No luck ...
>
> Any suggestion regarding conditions, etc.?
>
> This is the real situation:
>
> Insert A:  P~EcoRI ----  blunt~P
> Insert B:  P~BamHI ----  blunt~P
>
> Vector:   ---- EcoRI~OH       OH~BamHI ----
>
> Thanx in advance
> Dr. Hiranya Sankar Roychowdhury
> College Asst. Prof.
> Molecular Biology,
> Dept. of Chemistry & Biochemistry
> Box 30001 - 3MLS
> New Mexico State University
> Las Cruces, NM 88003
>
> Lab: (505) 646 4722
> Office: (505) 646 8256
> hroychow at nmsu.edu
>
> ---

Hello,

Are you getting no colonies after transformation? or you're getting "wrong"
plasmids.

I have tried few times ligation of three pieces (all sticky) and the efficiency
is low, I don't wonder if in your case this would be even lower such that you
have one blunt end.

Anyhow, how I have succeeded is in increasing the inserts:vector molar ratio (I
have made 3-5 different ratios, 5:1, 10:1, 20:1; and in every case got
different result). I have used the Promega's Ligafast and incubated several
hours (even overnight) in the fridge or 16 degrees. I am not sure if this
particular enzyme system makes a difference, it might be that other enzymes are
good enough. I transform with the whole ligation mix and after growing I plate
all the cells (pellet from a 1-2 ml culture). I have also included controls
either without insert A, or insert B or both. Usually I get only couple of
colonies per plate in the best cases, although it is just one what your looking
for, isn't it?

Can you find an enzyme for your vector such it gives you blunt end so you can
do the ligation in two steps? Can you PCR your inserts such that you can
introduce a restriction site for a sticky end generator restriction enzyme
enzyme instead blunt ends?
Other possibility, longer way but probably more successful, is to ligate the
inserts together. Even if the ligation efficiency is very low you may generate
enough to PCR the whole A:B insert. Then only digest and ligate with your
vector.

Hope this is of any help.

Good luck,

Arturo
_______________________________________________________________

Arturo Garcia-Horsman, Ph.D.
Research Specialist
Department of Pharmacology and Toxicology
University of Kuopio
Canthia, Harjulantie 1 A, 1. krs.
70210 Kuopio, Finland
Tel: +358-017-162422
Fax: +358-017-162424
E-mail: Arturo.Garcia-Horsman at uku.fi





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