can NOT subclone from pgemT-EZ into pmir-Report *help?* - (Aug/29/2012 )

Sooo.... after about a YEAR of trying this on my own, I am asking for help!

I am trying to ultimately get a couple of 3'UTRs, each of about 1000bp, into pmir-report.

1) I designed primers with appropriate engineered restriction sites (SacI-HF and HindIII-HF.)
2) I do the PCR, run in through a gel to clean it up and check the sizes.
3) Clone into PGEMT-EZ and transform in either JM109 or DH5-a competent cells.

A this point, I get nice colonies, so I....

4) grow up the colonies in liquid LB+amp, and miniprep them,
5) I digest the DNA with SacI-HF and HindII-HF in buffer 4 at 37degrees, then..
6) I run the digest on the gel.

I this point I have two nice bands for each digest, one at 3000 for pgemt, at ~1000 for the insert. Looks good so far.

7) So then I also digest pmir-report with the same two enzymes, run it on a gel to separate enzyme from vector, and to verify vector size.

8) I cut both the inserts and the vector out of the gel, gel-purify them, and try to ligate them together with T4DNA ligase and buffer.

9. Then I attempt another transformation, but have never, ever, ever had a colony with an insert, and very rarely do I get colonies at all! (even though I can grow pmir-report by itself with no insert.)


Any thoughts???

So, just to check that I undersand: you have a pGEMT vector with your desired insert. You have a pMIR-report vector with a (different?) insert. You cut both with SacI and HindI. They each give two bands on the gel, showing that they have been cut. You purify the pMIR-report vector backbone and the desired inserts, and then attempt to ligate and transform them. Is this right?

Here I explained how I do cloning (I do not say that it is perfect or it is the only way, but it works for me):
http://www.protocol-online.org/forums/topic/26650-isolation-of-plasmid-midi-and-maxi-prep/
I also explain why I do certain steps.
And here I advise on religations:
http://www.protocol-online.org/forums/topic/26683-subcloning-re-digest-and-ligation-and-transformation/

Hope this helps a bit,

Andreea

phage434 on Thu Aug 30 01:30:59 2012 said:

I have a pGEMT-EZ vector with one of two inserts, each of which are successfully digested from pGEMT. I digest the empty pMIR-report vector, and attempt to ligate and transform the insert from pGEMT into pMIR-report, with absolutely zero success.

I don't know if it's a problem with the digest of pMIR-report, or with the ligation, or with the transformation, but it has been a YEAR in the works....

ascacioc on Thu Aug 30 06:49:06 2012 said:

Hey! Thanks so much for the protocols. I will print them out and see if anything catches my eye.

~Ted

I'd check to see if you can digest pMIR-report with each of the enzymes individually. Since there is no insert, the only way to tell is by looking for the linearization of supercoiled DNA. Run three lanes with undigested, and cut with each of the two enzymes individually. You should see the disappearance of the supercoiled bands in the cut fragments. If one of the two enzymes does not cut, you have your problem. Do you have sequence for the plasmid at the cloning site? I'd recommend sequencing that region. I certainly would have done it by now. Your cut sites may be too close to one another to allow efficient cutting.