Hi
While investigating if recovery of larger samples of a DNA origami annealing reaction is possible with my current gel electrophoresis set up I discovered that according to thermo fisher
https://www.thermofisher.com/uk/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html
The maximum quantity of DNA that can be clearly identified in a band is 100 ng.
In my project Im currently using 5ul (1.12ug) of P7249 scaffold (Where the total weight is 112ug in 500uL) in a single annealing 50uL reaction and then a ten fold excess of staples (~11ug) even if I only use 5uL of product per lane I'm already in excess of 100ng by roughly ten fold (~1.2ug per lane).
I feel like 100ng of DNA per well is low? That would be 0.5uL of my folding reaction. If I recovered this volume I would be left with a 0.1 nanomolar solution of origami assuming recovery from a DNA extraction column in a 50ul buffer. Does anyone have experience recovering larger sample volumes?
I think you are looking at the wrong advice, the advice on Fisher website you linked is for the best resolution for your gel image (I.e. clear bands no leaking into neighbouring wells etc) not for optimal recovery of DNA product, most gel purification kits easily purify up to 10ug per column. The advice for optimal gel display and for optimal product recovery is different. For gels that I want to look neat and tidy I usually load a maximum of 5ul of product. If I was planning to excise the bands and carry out a gel extraction I would load all 50ul (in 2 neighbouring wells of 25ul each) to maximise your product recovery, you will likely get huge, intense bands and possibly leakthrough into neighboring lanes. Leave an empty lane between the ladder and your samples and also between each pair of samples if you are doing multiple gel extractions per gel.
Another factor to take into account is that the columns used in most gel extraction protocols have a maximum amount of DNA that they can bind. If your bands are extremely bright then you may overload the column. This has 2 solutions: 1) you can retain the flow through (which you normally discard) put it to one side, then once you have eluted the DNA from the column you can re-bind DNA from the flow through and re-elute (into a separate tube as this product will be more dilute). Or you can split the reaction in 2 and run it through 2 columns simultaneously.
Thank you Sam Amsbury.
I actually thought I should've contrasted the amount recoverable by an extraction column but didn't get round to it yet... Thanks for your advice that's very clear now.
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