Hi Weifei, When I was looking at Holliday junction annealing I found that the salt concentration was important. NaCl around 300-500mM improved yield as did MgCl2 at lower (100-200mM) concentrations. So, it might be worth trying higher salt.
Dave -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of ChenWeiFei Sent: 03 July 2015 16:14 To: [email protected] Subject: [ccp4bb] DNA RNA annealing problem Dear all, I want to get a complex of DNA-RNA-protein. But I have a big problem of annealing DNA-RNA. The length of DNA is 19nt and RNA is 17nt. Annealing protocol: 2uM DNA 2uM RNA 10mM Tris-Hcl 100mMNaCl 1mMEDTA Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature. I can just get a result of two single strand DNA/RNA. PAGE analysis. No double helix was founded. Does anyone have the same problem or know how to fix it. Thank you for your answering. Best regards, Weifei ________________________________ AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
