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Old 08-23-2019, 02:06 PM   #13
itstrieu
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Location: Atlanta

Join Date: Nov 2018
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Quote:
Originally Posted by mamor View Post
I haven't been checking the pH. It seems like the best place to start.
I do use calibrated pipettes, but I also have an addition dilution step that reduces the final concentration of NaOH to 0.4 mM. Does anyone have any thoughts on my process?

For denaturing and dilution:
- start with a 10 nM library and 0.2N NaOH (library: 10,000 pM, NaOH: 200 mM)
- 1:1 dilution with 0.2N NaOH (library: 5000 pM, NaOH: 100 mM)
- Add 990 uL HT1 (library: 50 pM, NaOH: 1 mM)
- Dilute library to 20 pM = 240 uL library:360 uL HT1 (library: 20 pM, NaOH: 0.4 mM)
Our workflow is a little bit different when we use the MiSeq. Usually it is the following:

1. Normalize all samples to 8 nM and pool them together.
2. Qubit the pool to see how far off it is from 8 nM and normalize to 4 nM.
3. Take 5 uL of 4 nM library + 5 uL of 0.2N NaOH. 5 minute incubation.
4. Add 990 uL of cold HT1 buffer to get to 20 pM.
5. Dilute 20 pM library to final loading concentration.
6. Add final library to a new tube with amount of desired PhiX. (i.e 540 uL of 16.5 pM library + 60 uL of 16.5 pM PhiX for 10% spike-in).

If you are worried about the final concentration of NaOH left, you can always add 5 uL of 200 mM Tris-HCl after the denaturation step and add 985 uL of HT1 instead of 990 uL.

Last edited by itstrieu; 08-23-2019 at 02:10 PM.
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