I run some plasmid samples with 1% agarose gel (like the picture shown), and the band is not clear,
I use 5ul DNA sample with 1ul dye in each wall, 120V for 40 minutes,
1 and 6 are ladder, the other are product of E. coli transformation,
3 is 2 dilute to 1/10, 5 is 4 dilute to 1/10,
I want to know why the band is not clear? and which part is supercoil ?
Besides, I do transformation with sample 7, and it failed, is that mean the DNA is not useful or I can adjust reaction condition to proceed transformation to get colony on agar plate?
The gel on the left looks like some technical problem with the gel (or the picture is out of focus) but can’t say why. Generally the lower band (but not the one at the bottom of the gel) should be the supercoiled band. I would guess that is what you see in 3,7 and 9.
No idea why you aren’t getting transformants from the DNA in 7, it looks just fine to me So probably an issue with the transformation
You would normally expect to see 3 or 2 bands in the uncut plasmid lane due to the different conformations of the isolated plasmid, this is affected by the quality of your isolation process... supercoiled, linear and open circular are the conformations respectively you see from the bottom to top in the gel image... because of their conformation they have different migration speed, so the supercoiled is the fastest because of less friction in agarose gel….
However, the problem is that you do not have a clear band profile in your gel, a poor quality gel, and maybe DNA samples, U-shaped bands… so you cannot decide about the band profile.
I recommend checking the quality of your gel and not exposing it too much to UV. In addition, your bands are U-shaped, therefore, set the voltage to the optimum and don't overload your samples, and make sure your gel has completely solidified, especially in the areas touched by the comb, and try to remove the comb gently.
Verify the quality and quantity of your plasmid using the Nanodrop. Make sure you don't use too much DNA (by volume) or DNA with excessive impurities for bacterial transformation. Besides, check the antibiotic you use for bacterial culture. Before transformation digest your plasmid with an appropriate enzyme to make it linear and check the size to see whether the band belongs to your desired plasmid.
Hi,
What is the origin of your samples? was it a transformation after ligation? A Gibson assembly? Also, I would recommend to decrease the agarose percentage so you can have better resolution of those large size bands and make sure the gel has solidified enough. Otherwise, it may give you those smeared-like bands. I wouldn't recommend analyzing miniprepped plasmid with electrophoresis directly without linearizing first because it can be confusing. I would rather do analytical restriction digestion/colony PCR to confirm the transformants because you linearize your plasmid and it's easier to understand. I hope it helps!
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