after re-isolation of vector, get single band - (Sep/22/2009 )

Hi,

I got a vector from a colleague and it looked contaminated with DNase (I diagnosed this based on info from other threads on this website). I decided to re-isolate the vector by transforming Ecoli with the uncut stock he sent me.

It worked and I recovered DNA with the miniprep kit (qiagen), but when I ran it on a gel I only get one band rather than 3 like normal. The band runs at the size the cut (linear) vector should run: when I cut the newly isolated vector with ECOR1 and used AlkP to clean up the ends, the band was the same size as the 'uncut' vector (though it runs more cleanly--less smearing).

I tried the ligation with the cut, phosphatased vector but it didn't work, so I tried re-isolating again from a fresh transformation and prep. I used both the original stock sent by my colleague as well as the stock i re-isolated that gave only one band. Both vectors transformed the cells, so both stocks must have intact (uncut) vector...but I got the same thing on the gel, one band. Possibly there is a faint second band (running heavier than the cut band) with one of the preps, but nothing like a vector 'typically' runs (from my limited experience I usually see 3 bands with an uncut vector). Any idea what might be causing this?

Thanks so much!

That is definitely odd. What size is the plasmid? How much are you loading? How thick is the single band?

You don't always see multiple bands when running uncut vector on a gel. The bands represent the different conformations of the circular plasmid (ie super-coiled, relaxed etc) and you may not be getting the vector in all of these forms from your prep, depending on how good your prep was.

As for your cut plasmid- if it is running the right size for linearised plasmid on a gel, then you would assume it has been cut......

When you say your ligation didn't work, what do you mean? No +ve transformants right??
Have you tried a variety of ligation conditions?

Do you include an uncut vector transformation control- to compare your number of transformants??

leelee on Sep 23 2009, 11:25 PM said:

Hi Leelee,

Thanks for your thoughts... Yes, I got no +ve transformants with any of my ligation products, but I did get colonies with the uncut positive control. I tried a range of insert:vector ratios (1:3, 1:1, 3:1, 5:1, and 10:1), two different T4 ligases (NEB and Fermentas), and two different batches of reisolated vector as well as the original degraded vector, two different types of chemically competent cells (one commercial, one home-made), and did the reactions with and without b-mercap. I used 15ng of vector and 2uL ligase in each 20uL reaction, and used fresh buffer containing ATP, leaving the reactions at 16C overnight.

I am confident that the insert has been prepared correctly and that the transformation step isn't the problem (since the uncut positive control yields colonies), so this leads me to think that my plasmid prep or the ligation may be the problem. I didn't try using a cut but not posphatased positive control to check that the ligation was working--maybe this sould be my next step?

georgiadave on Sep 23 2009, 07:42 PM said:

Hi,

Thanks for your reply...The plasmid is 5.6kb (it is the BR322 vector with a kan cassette already inserted), and I am trying to ligate it with a 300bp fragment. I tried loading different amounts of vector on the gel, but I typically use around 100-300ng. In the original stock I received, the three bands were present but a very bright blob of low MW DNA also ran in the lane. In the re-isolated vector it smears from the top of the gel lane down to the band, and the band is about the size of the linear vector.