Hello,
I have isolated a phage from a soil sample. I got a LTL somewhere around 10^9 pfu/mL. I did DNA extractions and got great DNA concentrations, always over 100ng/uL and the 260/280 ratio between 1.8-2.0 I then take 10uL and mix it with 4uL of loading dye and load in the gel.
When I run the gel, the bands do not appear. One time, the band appeared but then disappeared after ten minutes. It looks like there is a smear on the gel as well. It did this with 1% and 2% gel at around ~100V. My next step is to try with a 3% gel and ~70V.
What is the possible reason as to why this is happening? Also I did restriction digestion and no bands were formed.
Thank you in advance for your help.
show the pictures of your gels please..
dont run 3% gel its too thick for genomic DNA
lets talk...
Hi.
1)Do you see any illumination near the agarose gel wells? if so then may be concentration is too high to come out of the well. May be you can try diluting the sample and loading it Eg: you can take 2ul, 5ul and load. and use only 0.8%-1% gel.
2) We cannot sometimes trust the Absorbance reading.Might be concentration is low to be shown on well. Run a PCR with negative control and see if u get amplification.or you can measure the concentration using Qubit assay[If you have access]
All the best
Both Jaroslaw Krol and Gadicherla Ramya make good points. You are running the wrong % percent agarose, I routinely use 0.7% agarose if I am expecting anything over 1kb. At the percent you are running, large fragments will not enter the gel.
So try a more appropriate gel and look to see if you have fluorescence either at the top (which would be large DNA fragments) or at the bottom which would be small degraded DNA.
Remember that a phage genome can have a very wide range of possible sizes from as small as 5kb to well over 100kb
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