Repeated inserts ligated to my plasmid??

Hi all I ligated my small insert (37bp) into 3.5kb plasmid, I got colonies but when I check the sequence of my insert, I found more than one insert ligated as a repeats, some clone got three...

01 February 2019 4,050 6 View

How to get the exact number of the successful transformant bacteria???

07 August 2018 5,877 5 View

Anyone know a suicide plasmid that can be used to delete a gene in E.coli strain?

Anyone know a suicide plasmid that can be used to delete a gene in E.coli strain? I need to use a suicide plasmid to delete a gene in E.coli strain. I used one for gene deletion in Pseudomonas but...

11 December 2017 187 1 View

I found a mysterious thing after protein purification anyone can help?

Dear Dave I am working on a protein (around 125KD) and I have got some mysterious things regarding the purification so I need your help. The protein was expressed using pET28a vector in E.coli...

06 July 2015 6,863 18 View

How can I detect and measure the pyrophosphate PPi releasing from enzymatic reaction?

I have purified a protein which suppose to adenylate amino acid consuming ATP and releasing AMP and PPi, so to test the protein activity I have to measure the released PPi in the solution. Can...

08 September 2014 8,805 18 View

How can I add a DNA linker to a vector?

I am going to design two oligonucleotides (the size about 20 base) which can be annealed together forming two sticky ends then will be ligated to the digested vector with the corresponding enzyme....

03 April 2014 7,947 1 View

How much of EDTA-free protease inhibitor should be used when extracting protein?

I am going to extract protein from BL21 but I do not know how much of EDTA-free protease inhibitor (tablet from Roche diagnostic) should I use.

31 December 2013 9,992 1 View

Can someone help with flag-tag to the C-terminus of the protein?

Does anyone know how to add Flag-tag to the C-terminus of the protein? What is the principle of the procedure? I am planning to transform bacterial gene to mammalian cells and I have to check the...

31 December 2013 2,442 6 View

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 View

What's the concentration recommend for recombinant protein use in western blotting?

I'm currently working having problem with the concentration for recombinant protein use in western blot, the one been used is Recombinant Human CD19 Fc Chimera Protein-rndsystem which does not...

24 February 2021 8,514 3 View

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Hello, I am performing a western blot of my SARS-CoV-2 Spike-pseudotyped lentiviral particles. I denature my virus by adding concentrated virus to 2X Laemmli buffer at 1:1, then boiling for...

17 February 2021 4,844 4 View

Kinetics of Enzyme production in Recombinant E Coli?

Hello, I am trying to find kinetic information (such as rate of enzyme production etc.) on the expression of taq polymerase in E-Coli after inserting a recombinant plasmid. Any help would be...

12 February 2021 1,386 3 View

Is it possible to quantify recombinant protein in the supernatant using SDS PAGE ?

I would like to quantify a recombinant protein (with native signal sequence) secreted by Pichia pastoris using the SDS PAGE. Since my standard protein (plant origin) is not pure, I would like to...

07 February 2021 3,880 9 View

How to measure the concentration of a labeled protein with no tryptophan or tyrosines?

Hey I'm trying to measure the concentration of a protein I've purified and then labeled with Atto 647 flourescent dye. The protein has no tryptophans or tyrosines (although does have...

04 February 2021 4,482 4 View

How to prepare protein samples in EMSA assay?

Recently l was studying EMSA assay, but l didn't find any detailed information about protein sample. we can use two kinds of protein, including crude protein extracts and recombinant proteins. but...

02 February 2021 10,047 3 View

Difference between assessing EC50 in presence of physiological levels of human serum albumin and alpha1 acid glycoprotein vs. % human serum?

01 February 2021 1,825 1 View

Alpha synuclein lack structurw (i.e some of the helix part goes into turn ), any cause behind this?

I am trying to simulate alpha synuclein (1xq8.pdb) in GROMACS using GROMOS53a6 ff. I have run it for 200 ns. At the end of simulation alpha helix protein losses its helical structure. I am not...

31 January 2021 4,285 3 View

How to increase the lower detection limit of ELISA?

Hi all, I am trying to establish an ELISA method for the quantification of a recombinant protein injected into mice. In the meantime I am trying to make calibration curves with the purified...

27 January 2021 8,702 3 View