Re: [ccp4bb] RNA for crystallization
Thanks for all suggestions. I am going to P32-label the RNA and see if it runs as a single species on the gel and if not, I'll do the HPLC. Alex On Mon, May 20, 2013 at 11:16 AM, Eugene Valkov eugene.val...@gmail.comwrote: Hi Alex, If you do not have access to HPLC equipment, another alternative is gel-purification using a PAGE setup under denaturing (urea) conditions. This has the advantage of being fairly simple and effective for a range of transcripts, but you will need a fairly large gel tank system to get good resolution. Ke and Doudna 2004 review in Methods is an excellent starting point for learning about practical issues involved in the crystallisation of protein/RNA complexes and purification of RNA for this purpose. Hope this helps, Eugene On 20 May 2013 15:20, A K alek6...@gmail.com wrote: Dear all, I have a crystallization-related question. I am going to co-crystallize protein with RNA. I ordered a short (10 mer and 14 mer) RNA from Dharmacon but did not choose the purification option while ordering them due to the budget issues. How critical is to HPLC purify them before setting drops? Aren't RNA synthesis protocols reliable enough nowadays for such short RNAs? Thanks, Alex -- Dr Eugene Valkov Division of Structural Studies MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
[ccp4bb] RNA for crystallization
Dear all, I have a crystallization-related question. I am going to co-crystallize protein with RNA. I ordered a short (10 mer and 14 mer) RNA from Dharmacon but did not choose the purification option while ordering them due to the budget issues. How critical is to HPLC purify them before setting drops? Aren't RNA synthesis protocols reliable enough nowadays for such short RNAs? Thanks, Alex
Re: [ccp4bb] RNA for crystallization
Dear Alex, In my experience it is essential to HPLC purify short-RNAs for crystallisations as the impurities will hinder your crystallisation attempts. Best wishes, Salima Dr. Salima Nurmohamed Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive Oxford OX3 7BN, UK -- Kay Kendall Junior Research Fellow On 20 May 2013, at 15:20, A K alek6...@gmail.com wrote: Dear all, I have a crystallization-related question. I am going to co-crystallize protein with RNA. I ordered a short (10 mer and 14 mer) RNA from Dharmacon but did not choose the purification option while ordering them due to the budget issues. How critical is to HPLC purify them before setting drops? Aren't RNA synthesis protocols reliable enough nowadays for such short RNAs? Thanks, Alex
Re: [ccp4bb] RNA for crystallization
On May 20, 2013, at 7:20 AM, A K alek6...@gmail.com wrote: Dear all, I have a crystallization-related question. I am going to co-crystallize protein with RNA. I ordered a short (10 mer and 14 mer) RNA from Dharmacon but did not choose the purification option while ordering them due to the budget issues. How critical is to HPLC purify them before setting drops? Aren't RNA synthesis protocols reliable enough nowadays for such short RNAs? Thanks, Alex Dear Alex: The coupling steps are rather more inefficient than with DNA, so you may have some non-negligible N-1, N-2, .. contaminations, as well as potentially incompletely deprotected RNAs. If the RNA is involved in making lattice contacts, this might be problematic. However, crystallization is also a purification procedure, so you could always try it first and see if it works. In one case in our lab, having a contaminant like this present was absolutely essential for obtaining crystals. Gel purification is cheap and easy, and not too much of a burden if you can avoid getting skin cancer from the UV light needed for shadowing the RNA. A 20% acrylamide sequencing gel mix would give you the nucleotide resolution you might need. Good luck. Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA
Re: [ccp4bb] RNA for crystallization
Hi Alex, If you do not have access to HPLC equipment, another alternative is gel-purification using a PAGE setup under denaturing (urea) conditions. This has the advantage of being fairly simple and effective for a range of transcripts, but you will need a fairly large gel tank system to get good resolution. Ke and Doudna 2004 review in Methods is an excellent starting point for learning about practical issues involved in the crystallisation of protein/RNA complexes and purification of RNA for this purpose. Hope this helps, Eugene On 20 May 2013 15:20, A K alek6...@gmail.com wrote: Dear all, I have a crystallization-related question. I am going to co-crystallize protein with RNA. I ordered a short (10 mer and 14 mer) RNA from Dharmacon but did not choose the purification option while ordering them due to the budget issues. How critical is to HPLC purify them before setting drops? Aren't RNA synthesis protocols reliable enough nowadays for such short RNAs? Thanks, Alex -- Dr Eugene Valkov Division of Structural Studies MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
