Help me! No colonies! Problem with gel purification? - (Mar/28/2006 )
Hi everyone! Having a few problems....
I'm trying to clone promoter fragments into GFP expression vector (pEGFP-1).
I'm PCRing my promoter fragment (with added restriction enzyme sites) from genomic DNA using Taq, subcloning it into TOPO vector (with success, although not consistent!), isolating promoter fragment by restriction digest, running on gel, purifying with kit, ligating into pEGFP overnight and transforming and finding no colonies!!!!! I'm sure it's the gel purification that's the problem because when I ligate digested products that have been cleaned up (phenol chloroform) but not selected by gel purification, I get colonies, although I obviously have to screen a large number to get one with the correct insert in the correct vector. i don't know what is going wrong as thus technique was working perfectly for me 6 months ago...and now nothing!
what exactly is your gel purification protocol?
Try to run a gel with the DNA after your purification, then you'll see if there is degradation or may be no DNA at all.
Take a good look at the manual, may be you have to add isopropanol or not, is the pH of the first buffer incorrect, is your fragment too big....
And as aimikins asked, what is your protocol? May be we have the answer then.
Thanks, I've used both Qiagen and Invitrogen PureLink kits - I've tried running the purification product on a gel and it looks OK - I'm thinking maybe the restriction ends have been damaged in some way? I don't get it 'cos this was working so well 6 months ago. We relocated to a different lab and now it's stopped working! Maybe I should try purifying the fragment from a gel without eth bromide/UV exposure. I've heard that can help.... Maybe I could try beta agarase extraction instead of the kits - I seem to lose so much DNA with the kits aanyway...
If you've seen a big change, you might want to check the wavelength of the UV you are using to image the gels. 365 nm is pretty benign to DNA, while 305 nm can trash the DNA very quickly. You might not have noticed the change if you have moved.
how big is your promoter? which Taq are u using?
did u sequence your clones?
I am trying to clone 1.5kb from genomic dna but till now I was not successful.
we also had problems using quiagen kits. i heard Invitogen should be better...