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:[log in to unmask]] On Behalf Of ChenWeiFei
Sent: 03 July 2015 16:14
To: [log in to unmask]
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
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