I did alkali lysis of my bacteria and I got DNA concentration of 4000ng/µL, A260/A280 = 2.2 and A260/A230 of about 1.9 - what would be the expected contaminants and what shall I do?
Your sample contaminated with RNA, so treat it with RNase to get rid of RNA. The sample should be A260/280=1.8
Alkaline lysis is a good, but dirty method. That's one of the reasons they can still sell the columns. That said, it is outstanding for cheap screening of colonies.
Here's a shorthand of 1-2-3 Rapid Mini-preps
Spin 1.5mL culture 1’@10.000rpm.
Discard supernatant.
Add 100μL P1, Resuspend.
Add 100μL P2, 2’@RT.
Add 100μL P3, mix.
2’@14.000rpm
Pierce the lipid layer with the tip of the pipet and transfer 200μL supernatant to new tube.
Add 200μL isopropanol and mix.
Spin 5’@14.000rpm.
Wash with 70% EtOH, 5’@14.000rpm.
Dry pellet and resuspend in 50μL H2O.
Note: for low copy-number plasmids resuspend in 20μL H2O with RNAse.
P1: 50mM TrisCl, pH 8.0
10mM EDTA
100μg/mL RNAse A
P2: 200mM NaOH
1% SDS (w/v)
P3: 3.0 M KAc, pH 5.5 (pH IS important!!!)
pH is known to affect A260/280 readings. In particular, a shift towards alkaline pH increases the ratio. However, higher ratios generally do not indicate contamination. You may find these informative:
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
http://biomedicalgenomics.org/RNA_quality_control.html
Hi Shaimaa,
According to this attached NonoDrop Technique Bulletin, it says "Higher 260/280 purity ratios are not indicative of an issue" (see page 2), unless it is significantly high.
I think it could be due to RNA contamination. Try to use RNAase and see if you still have the same results
You should made an electrophoresis of 1 ul of your DNA. RNA will migrates faster.
May be your samples contain RNA ,treat your sample with RNase and check it A260/A280.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques