Cloning problem - Digestion, ligation and transformation (Jan/18/2005 )

Hey folks,
I need Help!! Please!
I have to put a 3.5 kB fragment into a 6.5 kB vector.
The vector has a Km resistance gene.
I use invitrogen chemically competent cells.
This is what I do:
I PCR amplify the Insert- I have an Nhe1 site and an Asc1 site that is introduced by PCR.
The same is true for the vector.
I then sequentially digest the vector and insert with ASc1 and then Nhe1.
Then I run the sample out on a gel;gel purify using Q biogene-gel purification kit. I transform using 5 ul of ligation reaction
these are the results:
NO DNA added: 0 colonies
No insert added: 0
NO ligase added: 0
and
1ul vector, 8ul insert,ligase, ligase buffer: 0
So, basically I did not get any clones.
I was wondering if it is absolutely essential to purify after PCR and before digestion?
Any help is welcome.
Thanks!

check your enzymes will cut when located at the end of DNA cassettes. If they don't add some extra bases on the end.

In addition, try inactivating your ligase as this can inhibit transformation efficiency.

Also, cleanup your PCR product after digestion if you don't already do this.
Finally, try altering your vector:insert ratio.

Hey
Thanks for the prompt reply.
I know that the plasmid is linearized-but I have no certain way of telling that the insert and vector has been double cut since the restriction sites are too close to the ends.
Thats why I sequentially digest the vector and insert so that the chances that both enzymes have cut are higher.
Do you know how I can inactivate ligase?
I do cleanup the PCR product after digestion.
I wanted to know if I have to do so before the digestion ?
I will try altering the vector:insert ratio.
Any other suggestions?
Thanks in advance!

I've found it extremely important to clean the PCR BEFORE digesting. Polymerases seems to sit on the neds blocking restrction enzyme access. Also the advice to add a couple extra nucleotides at the ends of your oligos is great. It seems to give the enzyme a good "seat" to cut seems to help and is absolutely critical for some enzymes (see the NEB catalogue for more info).

You can always do the (almost) sure-fire ligation into TA-TOPO vectors from Invitrogen and cut out your fragment from there if you continue to have problems.

Good luck!

i've heard nothing but horror stories concerning chemically competent cells. have you tried electrocompetent ones?

I suggest you to measure the efficiency of your competent cells

sometimes it works better if you first clone your PCR product in a pBluescript or pBK vector and put it in a second step in your vector