Hi all,
I am digesting with a double digestion XbaI and BamHi the plasmid extract of different clinical bacterial isolates to see if they have the same plasmid profile or not. In the small gel, I can't understand whether the isolate 1 and 4 (same order as they were loaded in the wells ) are different or not from the other ones since I can't see the second band (looking from the top of the well)..And what could be the largest (more fainted)band on the top of the samples? A fragment coming from the digestion or anything else? In the second gel (the bigger), again, I don't understand if the first sample loaded is different from all the other ones. Also, I can't see the shortest band in the plasmid extract samples 2 , 3 , 4 and 5 unlike the remaining samples, although they seem similar. On what it could depend?
Thanks
Dear Silvia,
in the first gel all four plasmids are identical. You have some undigested and partially digested molecules that you can see in samples 1 and 4. The band that you're seeing in the well are remains of bacterial chromosome in a denatured fashion. They appear when you over-lyse your cells or when you increase your re-naturalization time.
In the second gel, what you see is a bad polymerized gel, there's some kind of bubble that affects migration of samples 2 to 5, you can see that all the bands of these samples are distorted. If sample 1 is your control plasmid, what happens is the same as with the small gel, you have partially digested and undigested plasmid molecules.
Try to increase digestion time, or change the digestion buffer. As XbaI and BamHI had differences (star activity in different buffers). Add always a control sample that you know that is well prepared and should digest appropriately.
And when you prepare a new big gel, boil the agarose completely, till no small "jellyfish" is visible. Allow to cold, a little bit, before pouring it into the gel casting unit, to avoid any damage to the gel tray.
hope this helps,
Beatriz Sabater-Munoz
I agree with Beatriz. you can select a four base cutter instead of using 2 six base cutters and analyze on 2% gel- with a control.
hope this helps,
Vasudha
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