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Posted by: sara27 Nov 21 2009, 03:04 PM

Hey everyone,
I've been trying to do some cloning and I can't get this last part to work and its driving me crazy, especially since someone else did a very similar one in my lab and theirs worked without a hitch and using protocols very very similar to mine.

I have a vector that i already cloned in an insert that I PCR'd out of yeast genome and by site directed mutagenisis I inserted 2 restriction sites into my insert: PmeI and PacI. I didn't sequence it, but I separately digested my clone with each of the enzymes and since there are no other sites presents if it linearized for both I took it as a good clone. so now I want to use those sites to insert a fragment between them.

For my vector prep, I grew up the bacteria and extracted a good amount of DNA that looked of pretty good quality on an agarose gel. Since other people in my lab found that even though you can do a double digest of PacI and PmeI according to NEB, it doesn't work well (and I tried this once as well and it didn't work in my hands either). So now I start by digesting 10ug of my vector with 20 units of PmeI in 40ul reaction with the appropriate buffer and BSA. I've tried digesting times from 2 hours to 8 hours at 37 degrees. After the digest, I've often run a small amount on an agarose gel and it looks good. I then do an ammonium acetate precipitation and ethanol precipitation overnight and then the next day resuspend the DNA in water and go for digestion number 2: with 20 units Pac1 in 40ul with appropriate buffer and BSA at 37 degrees for 2-8 hours. I have also tested the enzyme on another DNA fragment, and it appears to be cutting fine. (since the 2 sites on my vector are so close I can't visually see a single vs double cut). After the second digest I also CIP it for 10 minutes at 37 degrees.

I also treated my insert prep in a similar manner, only I cut it out of another construct. However, I just received (which also didn't work in my hands) my insert from someone else's prep who recently successfully ligated it into their vector, using the same procedures as I am)

After the 2nd digestion I gel purify using a QIAGEN kit, quantify the DNA and set up my ligation. I've been using 0.1ug vector and adding the insert at a 5:1 ratio. Usually, when I have done cloning in the past there has been much bigger size difference between my vector and insert. Here, my vector is about 3.5kb and my insert is about 2.3kb...could this be causing my problem? anyway, I mix together the water, insert and vector in a total volume of 8ul and heat it up for 10 minutes and then i spin it down briefly and cool it down a little on ice before adding 1ul each of ligase buffer and high concentration ligase. The ligase is brand new and has worked for others in the lab, so I didn't feel the need to do a control for it. I thought it might be the ATP in the ligase buffer, so i spiked it with fresh ATP but still got nothing.
I'm getting absolutely no growth after transformation, not even on my background plate. I've also tried the same thing without CIPing the vector and still get teh same results. I just cant' figure this out so any help would be GREATLY appreciated.

THANK YOU IN ADVANCE!
and sorry it was so long

Posted by: HomeBrew Nov 21 2009, 04:25 PM

First, a few easy questions...

Do you know your cells are competent? How do you perform your transformation? What antibiotic resistance are you selecting for? Do you allow an expression period in non-selective media before plating? How big is your plasmid?

And finally, perhaps most importantly, how close together are your two restriction sites?

Posted by: sara27 Nov 21 2009, 04:58 PM

The cells are competent. I'm using a communal batch between my lab that were all prepared at the same time and they have worked for another researcher's ligations as well as my transformation of the original plasmid. For the transformation, I thaw a tube of 200ul of cells on ice for maybe 10 minutes. Meanwhile I spin down my ligation reactions and I transfer all of the cells to my ligation reaction tube. I let it incubate on ice for about 30 minutes and then I heat shock for 2 minutes at 42 degrees. I place them on ice for a few minutes after to cool them back down. Normally for amp resistance (which is what i am selecting for) I tend to plate the entire transformation right away. Since this wasn't working I did try to pre-culture them with LB medium without any drugs for 1 hour before plating a couple times but never got a different result.

My vector is about 3.5kb, and my insert is about 2.3 the ligation should result in a pasmid about 5.8kb.

There are not many bases between the sites. I can't remember exaclty how many since I did not design the system, I took over the project from someone else. I think there were maybe 5 bases. However, this should not be a problem because the exact same procedure was used successfully to make very similar constructs with other genes. I have talked to the person before who designed the experiment and she double checked that the primers had everything right and it was just like the other constructs which worked.

I too believe that my digest is the problem, but I can't figure out how to resolve it.

Posted by: phage434 Nov 21 2009, 05:04 PM

When you say that you treated your insert "in a similar manner" does that include CIP treatment? If so, then you will never get transformants, since your insert will not ligate.

Are you doing a transformation efficiency control? No transformants could just be bad transformations.

Just how close together are your cut sites? If they are too close, then one or the other enzyme may not cut.

You could try ligating your insert only, heat kill the ligase, and run a gel. The gel should show a ladder of multiples of the insert length. Similarly for the vector (before CIP treatment).