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Questions related from Qian Deng
I am currently performing a plasmid construction based on T4 DNA ligase. I first designed primers to incorporate identical restriction sites at both the 3' and 5' ends of the target fragment. This...
last month 9,823 1 View
Recently, I purified DnaA proteins from two different species via nickel-affinity chromatography. Based on their respective theoretical pI values, I prepared tailored purification buffers....
last month 3,406 4 View
If I want to construct a plasmid containing only the E. coli chromosomal origin of replication fragment and the pUC19 backbone with its antibiotic resistance, how should I proceed? I could first...
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I currently have significant doubts regarding the pH of the protein purification buffer. When purifying proteins, do I need to calibrate the buffer pH at the purification temperature? For example,...
last month 5,949 2 View
The gel electrophoresis results are shown in the figure. The control plasmid (~10,000 bp) displays the standard three bands, whereas my extracted plasmid (~2,500 bp) consistently shows multiple...
24 July 2025 209 2 View
Newly extracted plasmids and old plasmids (500 ng each) were run together on a gel at 160V for 30 min (Figure 1). Lane 1: old plasmid. Lanes 2-4: new plasmid. The results show that the new plasmid...
23 July 2025 8,613 0 View
The following two images show E. coli Topoisomerase I activity assay samples run under different electrophoresis conditions, with running parameters annotated. Figure 1: 2 V/cm for 12 hours;...
19 July 2025 8,978 2 View
I need to purify two helicases, both derived from E. coli. One is the replicative helicase DnaB, which is co-expressed with its loading protein DnaC on a dual-expression vector. The other is the...
15 April 2025 2,361 1 View
How can I obtain relaxed circular plasmid DNA? I previously attempted to generate and purify E. coli-derived Topoisomerase I (Topo I) based on literature protocols, but no relaxation activity was...
15 April 2025 7,549 4 View
I constructed two plasmids for DnaA expression: one with an N-terminal His-tag and the other with a C-terminal His-tag, and neither tag was cleaved during purification.The purification buffer used...
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