Gel band of bacteria genomic DNA - (Oct/30/2008 )
Being a novice in molecular work, so i appreciate if u all would give comments here.
I am trying to isolate a gene from 2 bacteria samples (Pseudomonas aeruginosa and Klebsiella pneumoniae).
After genomic DNA extraction, i performed gel electrophoresis to check whether the extraction was successful or not. The gel appeared somehow confused me. sorry for the quality because of my shallow experience. The 1% agarose electrophoresis performed at 120V for 35mins.
(Note: no DNA ladder was used here because i just wan to check the presence of DNA)
my questions are:
1. Is it what we called as "smear" of my band at upper there?
2. I wonder what are the two lower bands down there ? is it RNA since i didnt use of Rnase in my protol?
3. Is it possible to use that extracted DNA for further work, such as PCR ? because i would need to PCR the gene of interest out.
4. So what is the benchmark used to determine whether the quality of band is good enough for PCR later?
OK. Thanks. Hope to hear from anyone who read this. Thank you.
1. Centrifuge 1.5 mL of fresh cultures at 10000rpm for 30 seconds.
2. Centrifuge, resuspend in 567 μL TE buffer.
3. Add 3μL Proteinase K (20mg/mL) and 30μL 25% w/v SDS (0.1g/mL)
4. Add 100 μL of 5M NaCl and mix, add 80 μL CTAB/NaCl sol, mix. Incubate for 10 minutes at 65C.
5. Add equal volume of Phenol:Chloroform: Isoamyl Alcohol (25:24:1), invert several times till forms a single layer. Centrifuge for 5 minutes. Extract the clear supernatant into new tube. Repeat this step again.
6. Repeat step 7, but extract with Chloroform:Isoamyl Alcohol (24:1) once.
7. Add 0.6 volume isopropanol, invert several times to ppt DNA.
8. Centrifuge for 5 minute, ,decant supernatant, wash with 70% ethanol
9. Remove supernatant, air-dry the pellet
10. Dissolve DNA pellet in 100L TμE buffer and store at -4C.
That looks like big genomic DNA to me, but a marker would have been useful. When you repeat the gel, I'd try a 0.8% gel, and include a marker like lambda/Hind III.
Could be, but my RNAa often appears as more of a smear. Still, it could well be rRNA, I suppose.
Should be OK for PCR, your protocol looks OK to me.
Benchmark is an OD260/280 of ~1.8, or else a successful PCR.