Digestion and ligation problem - Insertion of 4Kb DNA fragment into a 10Kb vector (Jan/13/2006 )
I have a DNA fragment of 7Kb, with 3 genes of the fungi Aspergillus nidulans[/i]. This fragment is in a 10Kb plasmid (pRG3), so that the construction has 17Kb. When I digest this construction with BamHI enzime, I obtain 13Kb and 4Kb bands.
I want to separate this two bands, recircularize the bigest one and introduce the last one in the empty plasmid pRG3. So I digest the empty plasmid and the construction of 17Kb with BamHI. I cut the electrophoresis bands and purify each one with Geneclean kit. When I try to make ligations, I obtain no coli transformants. I use a ligation kit (all the kit I use are from Sigma, purification kit, ligation kit, the restriction enzyme...) with its positive control and it´s OK. The positive control for coli transformation is OK too. But I nothing about the desired constructions.
I know that the 13Kb fragment is probably too high for Geneclean purification (10Kb limit ) but the 4Kb and 10Kb fragments have good sizes. Furthermore I don´t know any other way to separate 4Kb and 13Kb fragments.
I also know that small pieces of agarose can inhibit the ligase, so should I precipitate and purify my samples before ligation?
Any other enzyme that someone think is good for this purpose?
Something that could help me?
Oier 778
I would think you would get many colonies on from no-insert vector-religation events, since there is no mention of phosphatase. this suggests to me that there is a problem somewhere between the gel and the ligation (probably your transformation protocol is OK since you are getting your positive controls?)
can you give more details about these steps, please? in particular, how do you do your ligations?
can you give more details about these steps, please? in particular, how do you do your ligations?
Thank you for answering.
I forgot the dephosphorilation step. Of course, I did it with the 10Kb empty and BamHI digested pRG3 plasmid. 200ng of digested plasmid were dephosphorilated with Shrimp phohsphatase from Sigma. 2 microlitres of this mix (about 20ng) were ligated with 100-160ng of the 4Kb insert. 1u of Sigma T4 ligase, 2 microlitres ATP (10mM) and 2 microlitres of buffer 10x were added to the mix, for a 20 microlitres final volume. I had no coli transformants after 16 hours ligation at 16 degrees in a water bath (I transformed 15 microlitres of the ligation mix).
Previously to this step, I separated this three fragments by agarose electroforesis and purified them by GenClean Gel Extraction Kit. And then the ligation. Nothing.
Furthermore, I tried to ligate 4Kb-10Kb fragment after purifying the 10Kb vector with clean-up columns (digestion with BamHI, purification with the columns, dephosphorilation and ligation with Gel Extraction Kit purified 4Kb insert). No coli transformants.