Help-loosing DNA after the cleaning - (Oct/29/2005 )

Hi, I will really appreciate if someone could help.
I try to create chimeric DNA from two products of PCR. I clean the products with Promega kit after every restriction and ligation and finally, when I want to ligate the new-created chimera into plasmid- there is no DNA remained (I afraid because of multiple cleanings).
I try to reduce the number of cleanings. Do I have to clean after the ligation of two fragments, before the restriction with enzymes (with which I also restrict the plasmid).
Thank you, Sem

Generate your PCR products, digest them and your vector (also CIP your vector), clean all three on one gel, combine all three and ligate in a three-way ligation. Screen transformants by colony PCR.

Well, if by "after the ligation", you really mean "after I digest with the second enzyme", then yes. But, I'm curious why you designed your experiment in such a manner?

I would have proceeded like this:

1st PCR product:

forward primer -- add a site for enzyme X to the 5' end
reverse primer -- add a site for enzyme Y to the 5' end

2nd PCR product:

forward primer -- add a site for enzyme Y to the 5' end
reverse primer -- add a site for enzyme X to the 5' end

vector:

Cut with enzyme X and CIP.

This assumes that sites for enzymes X & Y do not appear in either of your PCR products, and that the vector site for X is an appropriate cloning site in your vector. It also assumes that X & Y do not produce ends compatible with each other...

Can you do it that way?

The reason I don’t do so: I am afraid that exactly as after cutting two fragments (1 and 2) with enzyme y I expect they will ligate, the same will happen after cutting with x: the forward primer of 1st PCR product will ligate with reverse primer of 2nd PCR product.
The second reason: cutting the vector with one enzyme even using alcal. phospatase, you never know what happens. I will be very glad if you comment this, I am not a junior in this field. Thanks.

P.S. Of course I ment I am not a specialist, I am a junior

I understand what you're saying -- that in the ligation mix, the fate of the PCR-produced fragments will be thus:

A: X-fragment_1-Y--fragment_2-X
B: Y-fragment_1-X--fragment_2-Y
C: X-fragment_2-Y--fragment_1-X
D: Y-fragment_2-X--fragment_1-Y
E: X-fragment_1-Y--fragment_1-X
F: X-fragment_2-Y--fragment_2-X
G: Y-fragment_1-X--fragment_1-Y
H: Y-fragment_2-X--fragment_2-Y

But, you'll also have:

I: X-vector-X-fragment_1-Y
J: X-vector-X-fragment_2-Y

ocurring.

Now, when you transform, you'll select only for replicons (plasmids that can replicate). Ignoring re-circularized vector and vector-vector ligations (because you treated you vector with CIP), and further ignoring uncut vector remaining in your ligation mix (reduced dramatically because you gel purified your cut and CIP treated vector), you'll wind up with either:

vector + A
vector + C
vector + E
vector + F

And you can screen the colonies by PCR.

If you can cut your vector with two different enzynes that don't produce compatible ends, then I would proceed thusly:

1st PCR product:

forward primer -- add a site for enzyme X to the 5' end
reverse primer -- add a site for enzyme Y to the 5' end

2nd PCR product:

forward primer -- add a site for enzyme Y to the 5' end
reverse primer -- add a site for enzyme Z to the 5' end

vector:

Cut with enzyme X and Z.

Now the situation simplifies dramatically (assumming Y also produces ends incompatible with X and Z, and that your PCR products don't contain any sites for X, Y or Z).

But, I must tell you -- I clone via the first senario routinely, and though it looks on paper like you'd never get your clone, it works fine. We are often forced to cut the vector with a single enzyme, either because of sites within the PCR products, or because of the vector (some of the genetic tools we have available to us to work in the speices I study are pretty crude -- no MCS, no B/W selection, etc).

Look, the second scenario with x,y,z that is exactly the protocol I use, but because my x and y have compatible ends and may be ligated to each other, I am forced to do a number of stages, and loose the DNA. But I think I will try two things: 1) the first method you advised
2) may be the starting amount of DNA will be not 1 microgr. for each fragment but 5, what do you think?

Okay, I'm missing something here: Why must you use Nhe I and Spe I?

A couple of reasons: 1) these enzymes do no cut my fragments and vector in the middle 2) they produce a sticky ends 3) I have them in my lab