Laura Dionisi

12 Questions 18 Answers 0 Followers

Questions related from Laura Dionisi

Positive colony-PCR but plasmid sequencing shows no inserted gene?

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 5,015 9 View

Problem with cloning and plasmid mini-prep?

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 4,745 14 View

Control PCR to evaluate insertion of DNA into the plasmid?

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 7,026 3 View

Optimal LB broth salt concentration for E.coli DH5α?

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 309 4 View

Consequences of excess bases in the MCS after digestion?

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 2,942 4 View

How to scale up or down a restriction enzyme reaction?

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 1,190 3 View

How to set up a restriction enzyme reaction?

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 5,256 3 View

Degenerated primers for sequencing?

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 7,658 2 View

Reverse transcription PCR without probes?

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 3,313 5 View

What comes after ligation reaction?

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 9,284 7 View

Leader sequences to obtain secreted recombinant proteins ?

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 4,876 3 View

CDNA from a negative-sense RNA virus?

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 9,552 3 View