Does anybody know how to clone gene with one restriction enzyme Hind III?

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

How can we now if the promotor in the vector is well?

Hi, everyone. I want to transfer two genes in to the pseudomonas bacteria that isolated from the soil. The bacteria that I want to use for cloning haven't been identified and not be sequenced,...

02 August 2024 3,987 1 View

What is the relationship between protein structure and N or C terminal tagging choosing?

I want to do 2,3-butanediol dehydrogenase(BDH) enzyme purification to confirm its activity for 2,3-butanediol. Before that, I need to confirm which N or C terminal tagging is better for enzyme...

28 July 2024 366 3 View

Illustra™ MicroSpin™ G-25 columns what it is used for?

Hello, We found three packages of Illustra™ MicroSpin™ G-25 columns in the cabinet of an unused lab. They are very old but have never been opened. I have never used this kit before, and I couldn't...

25 July 2024 4,927 3 View

How can I calculate the SOD enzymatic activities using the spectrophotometer method?

I have to quantify the enzymatic activities of SOD in plants using a method that employs hypoxanthine-xanthine oxidase to create O2. The colorimetric reaction was performed by combining...

23 July 2024 8,492 3 View

Pink bacterial colonies?

I am cloning an overexpression plasmid with my protein of interest tagged with mScarlet. After transforming my ligated product into DH5α bacteria and plating on LB agar, I noticed colonies with a...

22 July 2024 5,953 6 View

What are the strategies to Enhance IgG-Producing Plasma Cells in mice for Monoclonal Antibody Development?

I have immunized BalB/C mice with a protein using the intradermal (ID) method with Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA), following a 14-day interval and three...

22 July 2024 9,160 2 View

Is there a method that I can measure alcohol dehydrogenase activity using NAD+ in enzyme extraction from plants?

17 July 2024 935 1 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...

17 July 2024 2,787 4 View

Isolation of Hydrogen Producing Bacteria?

any good research which can show selective microbial strains for producing hydrogen? Yes the normal procedure is known but the isolation is the key for such selective microbial strains which can...

13 July 2024 2,255 3 View

Kshitiz Singh

You can use Gibson Assembly to clone gene of your interest at the specific site. You need to PCR amplify your gene with primers having 20 to 25 bp overlapping arms at the site where you want to clone. Total size of each forward and reverse primer will be 45 to 50 bp containing Sequence corresponding to your gene (20-25 bp) + overlapping arms at site of cloning (20 - 25 bp). You can use Gibson assembly kit from NEB. This technique is very effective and efficient, if done correctly.

Gazmend Temaj

Dear Dr. Singh, many thaks for advices.

Sincerely yours,

Dear Gazmend,

If you need specific advises related to this. I will be happy to help.

Best regards,

Kshitiz