To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction

Abstract

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.

Mannose-modified erythrocyte membrane-encapsulated chitovanic nanoparticles as a DNA vaccine carrier against reticuloendothelial tissue hyperplasia virus

“…The pTT5-gp90 recombinant plasmid (Nanjing, Jiangsu, China) was prepared according to the plasmid extraction kit instructions and purified using PEG8000 following a previously published study ( 16 ). Briefly, 250 μg/mL of carboxymethyl chitosan and 100 μg/mL of pcDNA-gp90 plasmid solution were mixed, stirred for 30 s, and stored at 25 °C for 1 h ( 17 ).…”

The Influence of Variable-Heavy (VH) Chain Families on IgG2,3,4on FcγRs and Antibody Superantigens Protein G and L Binding using Biolayer Interferometry

“…All Pertuzumab V H sequences IgG 2 , 3 and 4 were recombinantly made as described previously [3] and joined with our previously Pertuzumab CDR grafted V H 1-7 [23] to make the respective recombinant V H 1-7 IgG 2-4 . Briefly, the inserts were PCR-amplified out with the adaptors and ligated, followed by transformation into competent E. coli (DH5α) strains [24, 25] and plasmid extraction [26] and transfected into HEK293 cells [3]. Transfection, production, and purification were performed previously [5, 27] using HEK293 cell lines transient transfection.…”

Development of qPCR Detection Kit for Langya Henipavirus L Gene

“…The L gene template sequence was obtained from GenBank (Accession number: OM101125.1), and the initial 540bp of DNA sequence in the L gene was selected as the template. The provided template was inserted into the pUC57 plasmid and transformed into Top10 Escherichia coli [14], followed by processing as previously discussed [15,16].The DNA synthesis was performed by Sangon Biotech (Shanghai, China).…”

“…Primer set 1 was designed by Sangong Biotech, primer set 2 and primer set 3 were designed using the Primer3Plus by ourselves [17]. To revive E. coli with the gene template from glycerol freezing, the glycerol culture was streaked on LB solid medium (Qingdao Hope Bio-Technology) without antibiotics [16]. After 24 hours of cultivation at 37°C, a single colony was selected and inoculated into 5mL of LB broth (Qingdao Hope Bio-Technology).…”