Kumar and Joe- I'm not sure if this helps but I always purified my oligos prior to crystallization. Originally, I would order them with the trityl-group on and purify then on a reverse phase HPLC column and used TFA to cleave on column. Although towards the end of the project, I just ordered them purified. I then would mix them together and anneal. For the project I was on, we had to try a bunch of different sequences that typically varied in the ends. I mixed purified protein and purified oligo together with a slight excess in DNA and set-up my drops. Crystals don't have to look good to diffract. Some of mine definitely did not, they looked almost like rice grains. Also, did you run a gel on the crystals to determine if both protein and DNA is in the crystals?
Mary On 7/16/07, JOE CRYSTAL <[EMAIL PROTECTED]> wrote:
Hi Kumar, I also have the same issue. If you get any helpful response, could you forward me a copy? Thank you. P.S. Could anyone who has any comments or suggestions on this issue also forward the response to ccp4bb? Thank you in advance. Best, Joe On 7/16/07, bputcha <[EMAIL PROTECTED]> wrote: > > Hi, > I am trying to crystallize a protein-DNA complex. I purify the protein > finally > using gel filtration. I purchase > single stranded complementary oligos (desalting from idtdna.com), mix > them up > and make DNA duplex by > heating to 95 degree C and cooling to room temperature. I mix protein > and DNA, > concentrate and use it > for crystallization. > I am geting small crystals consistently under a specific condition. > These > crystals take up IZIT dye but are > not well shaped. I am not able to improve the size and shape of the > crystals > substantially even after > screening with additives (Hampton research). > I suspect that purity of the duplex DNA (presence of unpaired oligos) is > > limiting the chances of obtaining > better crystals. > > How can I purify the duplex DNA further? > > Are there better ways of making protein-DNA complex for crystallization? > > If I make the protein –DNA complex and then do the gelfiltration, will > the > complex purified so be a better > choice for crystallization? > > Thank you > Kumar > > Dept. of Biochemistry, Cellular and Molecular Biology, > Walters Life Science, # 406, > University of Tennessee, TN, Knoxville, USA >
