RE: Denature Lambda DNA

[shankargan2006]

Hi,

I am working on a denaturing protocol to convert a ds Lambda DNA to ss DNA. Does anyone have a robust protocol using denaturing agents that can aid ds to ss DNA conversion, preserving streptavidin-biotin bonds? what’s the ideal pH and salt concentration to keep the single strand intact after the double stranded DNA was denatured?


Also, What is the largest commercially available Single Stranded DNA (that’s much longer than the M13 (7249 bp) DNA).

Desperately looking forward to your replies.

Thanks in advance.

Shankar

[moleculardude]

Hey there Shankargan,

I would use heat. This is the easiest, although you would need to cool the strands rapidly on ice from almost boiling.

Also optimize your protocol with NaCl and NaOH.

[drrico]

Shankar, my students routinely denature lambda genomic DNA to make subsatrtes for ssDNA exonuclease activities. We have had great success simply using a PCR machine to melt the DNA, followed by quick cooling on ice. If you keep the ionic strength low (10mM salt or less) it will help you preserve it in a denatured form.

DNA stability is compromised by fluctuations in downward, so include a buffer. 10-50 mM TrisHCl pH 7.5-8 is common around here.

The Strepavidin/Biotin interaction is Very Strong. Unless you boil the DNA with the avidin bound, you shouldn't have any trouble. I do not recommend denaturing the DNA with avidin bound to biotin....

NaOH is a good denaturant, too, and we used to use that to make templates for sequencing, but thermal denaturation in the PCR machine is our go-to method anymore...

[admin]

Great information there drrico!

thanks