When running an agarose gel with wells containing DNA and restriction enzymes, why is it that sometimes a few bands on the gel are higher than the index marker?
Such was the case for my control DNA well (no RE) and also for another well containing DNA and the restriction enzyme Pst I.
Please help.
2007-03-08 18:48:45 · 3 answers · asked by Anonymous in Science & Mathematics ➔ Biology
First off, your control DNA should just have one band. So if it didn't that means it somehow got restriction enzyme in there.
Second of all Pst 1 is a rare cutter and often used to digest high molecular weight DNA. So the real question, is are you digesting genomic DNA or DNA of known size? If Genomic, then you will definitely have bands of higher molecular weight than your marker. If it's a known size, what is the size? what is your marker size? It's possible that your Pst I is not digesting your DNA fully and the band is still the undigested DNA.
2007-03-09 03:18:43 · answer #1 · answered by btpage0630 5 · 0⤊ 0⤋
The index marker contain DNA fragment of known sizes...the smaller the size, the further the bands are fromt the wells...
Your control DNA above the marker is ok means it has an even larger size than the largest DNA fragment in the marker...
For your DNA with RE Pst I, it may be the product is also larger than the largest fragment in the marker...or it may just that you RE digest did not work, especially if the band of is as far as the control DNA...but it may mean that 2 Pst I sites are very near, cleaving away a very very small fragment...
Distance of band from well is proportionate to log10(size of DNA is bp)...
you can plot a graph from info derived from index marker and derive sizes of your sample DNA bands...
2007-03-08 18:58:28 · answer #2 · answered by lam_tensai 2 · 2⤊ 0⤋
Is the index marker really the lightest molecule in the sample? Or could there be a fraction which is lighter?
2007-03-08 18:52:59 · answer #3 · answered by Anonymous · 0⤊ 0⤋