clustering of very dilute libraries

[ps25]

Has anybody tried generating clusters from libraries that are already below 10nM? How do you denature the sample and maintain the number of clusters you are aiming for? If you add NaOH it dilutes the sample down even further. Has anyone tried using a lower concentration of NaOH or a different method for denaturation (eg heat denaturing)?

[hoisinjl]

We have loaded as low as 1nM libraries. We just scale the reaction. Take a 5nM reaction that we want to load at 10pM:

Load 6 ul of DNA (to denature at 1.5nM), 1ul of NaOH and 15ul EB buffer. Then dilute 6.7ul of the denatured library.

For 1nM it is slightly more complicated - 19ul DNA (denatured at 0.95nM), 1ul NaOH. The dilute 7.37ul for a 7pM reaction

[ps25]

Thanks hiosinjl. I just realised my typo mistake. Thats also what we do for libraries down to 1nM.
What I meant to ask is - how to dilute libraries that are below 1nM? If we just scale things at this point, the NaOH becomes too concentrated in the final dilution for clustering to be effective.

[BerkeleyGSL]

Neutralize the NaOH with HCl

We do this all the time, down as low as 10's of pM for some libraries. After the 5 min. incubation in 0.1N NaOH, we place the tube on ice and add an equal volume of HCl at the same concentration as the NaOH we added. Top up with the appropriate amount of cold hyb solution and you're good to go. pH is now neutral, just a little extra NaCl in there.

If you look at the Sanger method papers, they have a more complicated version for loading low concentration libraries if you look in the supplemental methods.

[ScottC]

Hi,

I'm just going through these calculations to make sure I'm doing this right...

Load 6 ul of DNA (to denature at 1.5nM), 1ul of NaOH and 15ul EB buffer. Then dilute 6.7ul of the denatured library.

Do you mean 6uL of DNA, 1uL NaOH and 13uL EB... or have I misunderstood?

Cheers,