2 replies to this topic

[joy123]

Hi! I extract genomic DNA (both human and bacteria, and I am interested in bacteria) from human liquid specimen, and plan to do qPCR with it.

The general DNA extraction procedure is: lysozyme-->protease K-->cell lysis-->protein precipitation-->centrifugation-->Isopropanol DNA extraction--> rinse with 70% Ethanol-->dry-->resolve with 10mM Tris (pH8.0).

The result turns that, the A260/280 is ~1.8. But the A260/230 is very low, ~0.9-1.4. The peak at 220 is high (attached).

I tried DNA precipitation with Isopronanol to get rid of salt, and resuspend in Tris, but the A260/230 is still low.

Then, I use sodium acetate+Ethanol to get DNA precipitaiton, and resuspend in Tris, but the A260/230 is still low.

My questions are:
1. Will it interfere with qPCR efficiency? Do I need to resolve the low A260/230?
2. Would you please give me some suggestions how to resolve this problem?

Thanks a lot!

Attached Thumbnails

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Edited by joy123, 27 August 2012 - 06:25 AM.

[AquaPlasmid]

The scans look normal to me. What were the DNA concentrations? Try use 10x higher concentrations.

[joy123]

The concentration is ~200ng/ul. I don't know what may caused the high peak at 220nm. And I wonder if it will interfere with qPCR assay.

AquaPlasmid, on 27 August 2012 - 04:58 PM, said:

The scans look normal to me. What were the DNA concentrations? Try use 10x higher concentrations.