Q1：Why is plasmid DNA yield low?
A1：
1) Plasmid copy number is low. Vectors can cause significant fluctuations in plasmid yield due to differences in copy number. High-copy-number vectors often fluctuate by 2 to 3 times the yield (3~16 μg of high-copy-number plasmid vectors per milliliter of bacterial culture cultured overnight). Long fragment plasmids and expression vectors are often dominated by medium and low copy numbers, and the yield per milliliter of bacterial solution is about 0.5~2 μg.
l Low-copy plasmids: pBR322, pACYC and its derivatives, pSC101 and its derivatives, SuperCos, pWE15.
l High copy plasmids: pTZ, pUC, pBS, pGM-T.
2) Bacteria problem. Plasmids are lost in the process of strain preservation. It is best to streak and activate before culturing bacteria to stabilize the yield.；
3) Bacteria are not sufficiently lysed. Bacteria must be fully resuspended in Buffer P1/RNase A, as agglomerated bacteria cannot be lysed, which will reduce the yield..
4) The reagent preparation is wrong. If there is precipitation in Buffer P2, it needs to be heated to dissolve, and the volume of ethanol added to Buffer W1 is not accurate.
5) The elution efficiency is low. Preheat the Elution Buffer to 60~65℃, and perform the second elution.
Q2：Why is there genomic DNA contamination in plasmid DNA?
A2：
1) 1) The bacterial culture time is too long. Bacterial liquid culture time should be controlled within 12~16 h.
2) 2) The cracking problem. When adding Buffer P2, it must be mixed by gentle inversion; when processing multiple samples, the total time should not exceed 5 min after adding Buffer P2.
