How to investigate the low yield of high copy plasmid small extraction kit

If you want to load a larger amount, it is recommended to directly switch to a miniprep or midiprep column labeled for high loading capacity, such as a 30-50 μg capacity or a dual-column membrane product. In terms of operation, the bacterial culture can also be enriched by centrifuging it in two batches into the same tube, but P1 resuspension must be thorough; otherwise, residual cell wall material will reduce lysis efficiency. Another common pitfall is antibiotics and culture medium: ampicillin degrades easily, and overnight culture will produce a large number of plasmid-free satellite bacteria, so the OD appears high but the plasmid proportion is low; for pUC19, it is best to use freshly transformed single colonies, shake for 12-14 h, and harvest bacteria at an OD600 of about 1.5, which is more suitable than 2.0. First fix the harvest time and bacterial culture volume before comparing brands; otherwise, the data cannot be attributed.

Longer lysis time is not always better. Alkaline lysis for more than 5 min will cause irreversible breakage of genomic DNA, which instead contaminates the plasmid and also prevents large fragments from being precipitated cleanly. It is recommended to strictly control the process: after adding P2, invert until clear, add N3/P3 within 3 min, place on ice for 5 min after adding the neutralization solution, centrifuge at high speed for 10 min, and take only the clear supernatant. Before elution, preheat the elution buffer to 70°C, add it to the center of the membrane, let it stand for 2 min, and then centrifuge; this can significantly improve recovery. If the Nanodrop reading is 80 ng/μl, also check A260/280 and A260/230. If the ratios are also low, it may be protein or guanidine salt residue, not a loading capacity issue.

The load capacity of the column can be referred to in the instruction manual. Common small lifting columns have a load capacity of 20-35 μ g, while some brands have a load capacity of 50 μ g. Under normal conditions, pUC19 can reach 5-15 μ g per ml of bacterial solution, and theoretically, 3 ml should be able to extract 15-40 μ g. Your actual 4 μ g indicates that the loading has not reached its limit, but rather that the lysis or binding has not been done properly. In terms of brand differentiation, Qiagen's spin miniprep has a stable but expensive loading capacity; Domestic Tian Gen, Nuo Wei Zan, and Kang Wei Xiao extraction columns can often achieve high copy yields of 15-25 μ g, pay attention to whether the lysate is clear. You can compare two brands using the same batch of bacterial solution at the same time, don't just change the washing volume.

PUC19 high copy, when OD600 to 2.0, the 3 ml bacterial volume usually exceeds the lysis limit of many small extraction columns. The nominal yield is often achieved using young bacterial solution with OD600 1.0-1.5. In your case, if you first reduce it to 1.5 ml of bacterial solution and keep the other conditions unchanged, the yield often increases instead. It is also possible to split 3 ml of bacterial solution and pass it through the same column twice, but it is more secure to split it into two columns. Your 80 ng/μ l × 50 μ l only has 4 μ g, which is significantly lower. First, rule out incomplete resuspension: there should be no visible bacterial mass after adding P1, and then lyse.