I run gel electrophoresis to confirm no presence of DNase contamination however my samples are remaining at the start of the gel. positive controls move as expected but my negative controls and samples both remain at the start point. I have remade wash buffers and carrier buffers but am still not seeing my samples move.
I faced this problem several times. This problem is due to the electrical current passage. Please, check your buffer.
Hi,
which kind of samples did you use? And what was the %age of the gel?
Maybe your negative samples can`t move into the gel due to size/complexity or anything else...
Did you perform the same experiment before?
Hey,
I agree with Radu, stacking gel is a huge help. Be sure to run the gel slower through the stacking gel, then you can increase the voltage through the separating gel. Ensure the stacking gel is about an inch or two long (not including the combs) to allow the proteins to separate appropriately.
For me it looks like high-molecular DNA or maybe plasmid DNA of great size. Are the positive controls in lane 4 and the last lane? What kind of samples/controls did you use and how did you treat them?
Since the DNA did not run that far, a lower %age of the gel could help (0,5 - 1%) for better separation. But for lower %ages I would recommend to use lower voltage to avoid to much heat.
what size is your DNA and what size standard are you using?. That looks like a good result to me with high molecular weight dna running above the size standard as expected but it would look better run in 1% gel and run at a lower voltage to improve the size standard as Yvonne says and if the very bright band is your dna then run a lower volume of sample. What is sample 6 and the similar running sample ?
hi thanks all for the help, positive controls are lane 5 and the last one, the first four signals are negative controls. the negative controls and unknown samples normally move further through the gel than the positive controls.
I have inherited the test from someone who left quite quickly and dont know too much about the background, but it had all been working as expected. The lab has been fully cleaned and most reagents changed as i was thinking of contamination given the negative controls are in line with the samples but this still has not produced a better result.
I may be missing something here but if the positive control has dNASE present then the dna will be smaller and so will move more quickly through the gel as lane 5 and the last one have done. The uncut higher molecular weight dna is running slower. If my understanding of your assay is correct then the gel is running correctly.
Like Paul, I would also assume that the positive controls have been treated with DNase and should therefore run further through the gel than the untreated negative controls. As your samples move the same way like the negative controls this should mean they were not contaminated with DNase. Therefore I agree with Paul that the gel is running correctly and you did successfully confirm no presence of DNase in your samples.
What I am wondering about is why you expected the other way around? If I understood everything correctly that wouldn't make much sense, or?
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