I performed a miniprep of a plasmid containing an insert of ~1000 bp (total size of construct
7712 bp) and loaded ~650 ng of this miniprep DNA into each well of the below gel.
I am worried that the bands are too fuzzy and that there is too much smearing on the gel indicating a poor quality miniprep. Or is this smearing simply from running too much DNA on the gel?
Would the plasmids from these minpreps likely be okay for transfecting HEK293T cells?
Yes, the bands are fuzzy because of heavy loading. Aim to load about 40 to 100 ng to see the bands clearly without streaking or saturating the gel.
If you are planning to transfect this construct, it is good practice to take about 1 to 2 ug aside and double digest it to release the insert. That way you can validate your construct from the size of the backbone and the fallout.
Checking the plasmid profile alone is done to confirm that the construct you have isolated exists mostly in the supercoiled form, which is ideal for transfection.
Yes, the bands are fuzzy because of heavy loading. Aim to load about 40 to 100 ng to see the bands clearly without streaking or saturating the gel.
If you are planning to transfect this construct, it is good practice to take about 1 to 2 ug aside and double digest it to release the insert. That way you can validate your construct from the size of the backbone and the fallout.
Checking the plasmid profile alone is done to confirm that the construct you have isolated exists mostly in the supercoiled form, which is ideal for transfection.
Thank you so much for the advice Praveesh Valissery
I re-ran the samples at around half the concentration and they came out looking much better (lanes 2 & 3). I also performed a double digest and the insert came out at the correct size (lanes 4 & 5). (Lane 1 is a 100 bp ladder.)
Would it be safe to say that there is no genomic DNA contamination in the plasmid samples run in lanes 2 & 3?
Ruth Purcell It looks good. There isn't any gDNA there. The upper bands near the well are probably nicked plamid. All the best with your transfection.
I agree with Praveesh Valissery that your minipreps look just fine, uncut plasmid always looks a bit odd due to the various different bands and your gel was overloaded. Your plasmid should be good to use.
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