Cloning/Miniprep Blues - High molecular weight bands iand no plasmid n my miniprep (Jun/23/2007 )
Hi I'm hoping I could get some help troubleshooting a problem i'm having with my miniprep. When I perform the alkaline lysis miniprep, I obtain DNA bands that are greater than 10kb, whilst the expected size of my recombinant plasmid is 8.4 kb. Furthermore, when I digest the extracted plasmid to check if they are recombinants, I obtain intense smears. The cells I cultured and used in miniprep have both ampicillin and kanamycin reisistance, as I am expecting them to, if they took up any plasmid at all. Therefore I should be isolating plasmid when I perform the miniprep. My competent cells (DH5 alpha) are pure and not contaminated. I know this because the negative control in transformation yielded no colonies on my ampicillin plate. The media used to culture the cells was sterile as an aliquot which was left uninocluated and set to grow along with the cultures showed no growth. The only thing I can pick at is that I'm doing something wrong during my miniprep, and this is what I need help sorting. I made fresh miniprep solutions and obtained the same distressing result that I described above. I have been doing miniprep for many years and this is the first time I am encountering such problems, and frankly I dont see what I could be doing so wrong during miniprep that would cause me to isolate such high MW DNA (which I think may be bacterial genomic DNA) and no plasmid at all. So now I doubt taht the problem is with my miniprep, but I dont know what to pick at now.
I hope u guys can offer some suggestions as to what I could do wether it be with the miniprep or the cloning procedure.
Hopeful Ann
What type of plasmids are these? Do they have any repeats?
We have noticed that with some viral vectors having inverted terminal repeats, they can undergo recombination and the DNA sizes vary upon purification.
We have noticed that with some viral vectors having inverted terminal repeats, they can undergo recombination and the DNA sizes vary upon purification.
The plasmid is pCR2.1 and I am trying to insert 1.95 copies of a 2.6 kb plant virus in it. I got the 0.95 fragment (approximately 2500 bp) in, and that construct is now the vector. I am trying to insert the full length virus (2600bp) now and I am getting these weird results after miniprep of my putative clones.
I was attempting miniprep of cultures of other cells I picked from the plates. The bacterial pellet from the cells have a reddish hue and one culture actually went green. Both these coloring is quite strange to me. I didnt bother with the miniprep, I'm just gonna start over. But does anyone have suggestions as to what may have happened? Especially to produce these colored bacterial pellets. My negative control from the ligation behaved properly, ie did not grow and my positive control is fine, ie lots of colonies that produce plasmids of expected size. I am gonna prepare the vector and insert again and repeat the cloning. Do you think I should make fresh competent cells or even try another E coli strain?