12 Questions 18 Answers 0 Followers
Questions related from Laura Dionisi
Good morning everyone, I am currently working on producing recombinant proteins. After trying several strategies with the classical cloning approach, I decided to use the Gibson Assembly...
07 August 2025 5,004 9 View
Hello everyone, I need some advice. I am working with recombinant proteins, and at the moment I am trying to insert a ~1 kb gene into a ~3000 bp plasmid (pT7-CHis). I performed a Gibson assembly...
25 June 2025 4,733 14 View
Hello, everyone, I am setting up the digestion and ligation reaction of a plasmid and DNA insert to be transformed into E. coli. Before proceeding with the transformation I wanted to verify the...
12 December 2024 7,012 3 View
Hi everyone, I noticed that there are different LB broth (lennox, miller) which are different in their sodium chloride concentration. As I am working with E. coli strain DH5α, will the choice of...
20 September 2024 298 4 View
Good morning, I am trying to produce a recombinant protein. Unfortunately I one of the two restriction enzymes that I use do not cut at the end of the MCS. After the digestion 15 nucleotides of...
18 July 2024 2,912 4 View
Hi I am trying to set up the reactions with my plasmid, insert and RE. Usually the standard final volume is 20 uL or 50 uL (it depends on the protocol provided by the manufacturer) with 1 ug of...
29 May 2024 1,167 3 View
I'm a doctoral student with early experiences with recombinant protein synthesis. Since I still have to start my experiment and I do not yet know the quantities of DNA and plasmid that I will...
20 May 2024 5,231 3 View
Hi My team and I are trying to design degenerated primers in order to sequence a viral genome. We do not know how many degeneration we can include in each primer. Is it possible to add up to 6/10...
23 February 2024 7,646 2 View
Hi, here in my lab I have a kit for RT-PCR with probes (for the real time quantification). Can I run my reaction without adding the probes, in order to obtain en “end point” product? I was...
01 February 2024 3,304 5 View
Hi, I have to perform a ligation reaction between a PCR fragment and a plasmid, in order to transform competent E. coli strain to amplify the plasmid. I will also make a gel to check the correct...
30 January 2024 9,278 7 View
Can anyone advise me on leader sequences to obtain recombinant proteins that are secreted by the cell culture? I should use mammalian cells for expression (from the Expi293 kit by ThermoFisher),...
09 January 2024 4,865 3 View
Hi! Has anyone worked with negative-sense RNA viruses? I need to express recombinant proteins using a vector, so it will be necessary to start with ssRNA(-) to obtain cDNA for selecting the gene...
02 January 2024 9,541 3 View