Near diploid human cancer cell line?

I am currently looking for a near diploid human cancer cell line, such as HCT116 and U87-MG. Preferably XY with modal chromosome # between 40-50, "normal" DNA repair pathways, and able to easily...

06 July 2015 4,638 2 View

Has anyone had an issue with Gibson Assembly not connecting a plasmid and a single insert?

Hello All, I am currently struggling with getting a Gibson Assembly to work. I am trying to insert a 2.5 Kb insert into a roughly 12 Kb plasmid (modified pLEX-MCS). I have had the G.A work in the...

11 December 2014 4,217 7 View

Has anyone encountered issues with MCF10a not incorporating BrdU?

Hello All, I am trying to do a simple fluorescence plus Giemsa stain/acridine orange differential stain for chromosomes. I have had great success in the past using MEF/CHO/and various cancer cell...

11 December 2014 837 2 View

Nonspecific PCR products and faint bands with Q5 polymerase - any thoughts?

Afternoon everyone, I am currently using Q5 polymerase from NEB to add restriction sites to an insert in a pBabepuro plasmid (~5400 Kb vector and 2.2 Kb insert). I have designed the primers to...

09 October 2014 4,493 4 View

Does anyone have the sequence for the pBabepuro-MYC-ER plasmid?

08 September 2014 1,422 1 View

Western Blot staining issue, poor B-actin band

I have been running several western blots over the past few days and have had the same issue where large proteins seem to stain nicely where as the smaller (~50 Kda) stain very poorly. I am using...

05 June 2013 9,720 4 View

How to calculate the fraction metabolized of a drug by an enzyme using clearance(ml/min) values of the metabolite formed?

I am trying to find the fraction metabolized of a drug by CYP and UGT enzymes. I am unable to get the values of fraction metabolized (Fm) directly from the literature. Can we calculate the Fm of a...

21 February 2021 5,379 2 View

Sanger Sequencing results not matching experimental results of Restriction Digestion, colony PCR and protein expression?

I have cloned two domains of a gene into two bacterial vectors using cut and paste cloning with different RE sites. I checked the putative clones by colony PCR and Restriction Digestion (fallouts...

18 February 2021 6,736 4 View

How long does it take to design and validate transition state analogue inhibitors?

Hi, I'm asking from the point of view of designing a research plan: How long (approximately) can it take to determine the transition state structure of an enzyme substrate using kinetic isotope...

14 February 2021 2,610 1 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

No title

Hello! I want to determine the activity of my in house Reverse Transcriptase enzyme. For this, I have used Thermo's Enzcheck Reverse Transcriptase assay kit. I used commercial Thermo RT enzyme to...

08 February 2021 7,285 1 View

How define one baker unit(BU) is the amount of hydrogen peroxide catalase ?

07 February 2021 2,006 1 View

Does DNA A-tailing survive after agarose gel electrophoresis and purification?

Hi everyone, I need to clone in pGEM-T vector the result of amplifying a metagenomic library by PCR with a high fidelity polymerase but I only want to clone the fragments of an specific size...

04 February 2021 6,051 8 View

Why RFLP Digestion looks in this way?

01 February 2021 9,576 3 View

Can any one provide information about medicine, epidemic and critical issues or it is mandatory to be an expert ?

Due to the pandemic of coronavirus, the most source that the people depend on was social media. There is no guaranteed solution that is biased from political opinion. An example of that the...

01 February 2021 7,567 3 View

Can I use 500ng of DNA for restriction digestion?

The recommended amount of DNA for restriction digestion is 1ug in a 50ul rx I've got around 500-600ng of DNA in a total volume of 35ul. Would I get satisfactory results with this amount of DNA for...

31 January 2021 729 6 View