Creating empty vector by self-ligation - (Apr/06/2009 )

I am attempting to create an empty vector from a commercial plasmid pCMVSport-6 (4.4kb) with Prp insert (around 2.5kb).
Therefore i carried out a double digestion on the pCMVSport6-Prp using Not1 and Sal1, incubate at 37 degree celcius for 2 hours.
Then i run the DNA on 1% agarose gel and here i can see two band. One is the vector backbone(4.4kb) and another is the insert 2.5kb.
I cut out the 4.4kb band and performed gel extraction. After extraction i run the DNA on gel, i was able to see one 4.4kb band.

The DNA eluted in TE buffer was then religate as followed:
DNA: 10ul
T4 ligase: 1ul
Buffer: 2ul
Deionized water: 7ul
total 20ul
incubate at 16 degree celcius for 1 hours and heat inactivate it at 65 degree celcius for 10 minutes.

5ul of religated DNA was then transformed into 100ul competent E coli DH5aplha.
But i can't get any colony and i would like to know where the problem lies.
for those who know, i would appreciate it if you could help.

Hey,

Why do you need to religate the vector, if you want to clone something else in sites which are present in the clone that you already have, just digest with those enzymes and proceed; why do you want to go a step back?

Besides, just check if Not I and Sal I are compatible.

Best,
TC

Not I and Sal I are not compatible. The ligation will not work. You will have to revise the strategy. Ligating an oligo containing ends which are compatible to SalI and NotI would solve the problem.

TC does raise a point. Is creation of a blank plasmid really necessary?

perneseblue on Apr 7 2009, 12:16 PM said:

You could also fill your sticky ends with Klenow, and then follow to ligate a blunt ended vector, which should work quite nicelly.

I'm assuming you want an empty vector for some transfection controls, if not... I add to TC and perneseblue, is it really necessary?

Actually I need the empty vector as tranfection control.
and how can i check the compatibility of Not 1 and Sal1?

by the way thanks alot for helping.

T C on Apr 7 2009, 02:33 PM said:

T C on Apr 7 2009, 02:33 PM said:

Actually I need the empty vector as tranfection control.
and how can i check the compatibility of Not 1 and Sal1?

by the way thanks alot for helping.

Hey,

By looking at the overhangs generated after the cleavage reaction, they should match: A should match with T and G with C in the overhangs...its fairly simple. Look at any standard text book and it will tell you.

Look at this:

http://bio.freelogy.org/wiki/Restriction_Digestion

Once you know how to do this, use standard tables freely available on the net to know about the compatibility in a matter of seconds. Also, some enzymes are Isoschizomers.

BTW Not and Sal I, as perneseblue rightly points our, are not compatible

Best,
TC

chijing on Apr 8 2009, 09:09 AM said:

perneseblue on Apr 7 2009, 07:16 PM said:

Do i have to buy oligo with ends compatible to Sal 1 and Not 1 in order to do this?

chijing on Apr 7 2009, 08:14 PM said:

Yes, you do.

How about this:

Find a site at the end of the gene that is already cloned in the vector or at the beginning of it , which is also present in the MCS of the vector, just splice the entire region out using this site and religate. That should work......but never tried it.

Best,
TC

chijing on Apr 8 2009, 10:44 AM said: