John Edward Simons

10 Questions 2 Answers 0 Followers

Questions related from John Edward Simons

Can I use GoldenGate assembly for library creation?

Hey all for the last few months I've been using GoldenGate assembly now construct a large mutant library for a directed evolution experiment. I'm not having success with this and the colonies I do...

30 May 2023 3,304 0 View

Promoter and terminator considerations for gene integration into the genome of E.coli?

Hi all, I am integrating a two gene pathway into the genome of E.coli. I have a good locus in which to integrate this and I have selected a good promoter. However, I am wondering do I need to...

11 February 2022 7,227 1 View

What's a good protocol to carry out Lamda red recombination into the E.coli genome?

Hi all, I'm working on this topic alone and would love some help and guidance if possible. I'm working from this paper - https://journals.asm.org/doi/10.1128/AEM.00313-14#B3 (High-Efficiency...

03 January 2022 387 2 View

Assembling ePCR library using Gibson assembly or Golden Gate assembly?

Assembling ePCR library using Gibson assembly or Golden Gate assembly? Currently I am using a GeneMorph kit to generate error prone PCR libraries of a gene region. Since the region I am using is...

27 October 2021 1,737 1 View

Using Gibson assembly to clone fragments generated by error prone PCR?

Hi all, I am carrying out an error prone PCR (ePCR) on a region internal to a gene that I cannot use restriction sites for. Therefore I cannot use restriction cloning to construct the library. I...

08 September 2021 5,579 1 View

Could anyone recommend a good yeast integration vector for protein expression?

Hi all, I am new to the world of yeast expression so forgive the basic question. I am looking to integrate a gene for a protein of interest into the yeast genome. What vector would you advice me...

14 June 2021 9,096 1 View

What's the best vector to use for stable integration of a gene into Saccharomyces?

Hi all I need to place a gene under the control of a galactose promoter in the yeast genome. The gene I need to place under the control of the gal promoter will also be a selection marker gene....

09 June 2021 5,785 3 View

What's the most efficient way to swap regions on a plasmid?

Hi everyone, I am looking for advice on how to swap regions on a plasmid I am working with. Basically I want to remove the Amp resistance gene from under the control of the Amp promoter and move...

17 May 2021 2,043 5 View

Basic question about cloning into a pET28a vector?

Hi all, This will be a stupid question but forgive me as I haven’t got much cloning experience. I want to clone a gene of interest into the pET28a expression systems but I’m confused. On the...

12 May 2021 4,402 3 View

How would I clone fragments generated by error prone PCR?

Hi all, This may be a stupid question but I want to generate a library using error prone PCR. Obviously I know the starting sequence but as the libraries can be completely randomised I'm not sure...

13 April 2021 2,704 3 View