Mutagenesis PCR Variable Yields Despite Exact Same Conditions???

Hello fellow scientists, I could really use some advice! I'm using a new(ish) Bio-Rad T100 thermocycler to run a mutagenesis PCR using NEB's Q5 Site Directed Mutagenesis kit (E0554S). My PCR...

15 November 2020 2,828 11 View

Grounded theory or Interpretive phenomenological analysis? Which is best for dissertation studying perinatal mental health?

I am curious what the differences are between using grounded theory or interpretive phenomenological analysis (IPA) for my dissertation. I am seeking to both complete my dissertation for...

29 October 2020 3,066 4 View

AlphaLISA SureFire assay troubleshooting - issues with phosphorylated protein detection?

We are currently working with a fluorescent AlphaLISA assay platform and are experiencing issues with detection of the phosphorylated protein of interest, despite numerous attempts at...

23 June 2020 7,118 2 View

Best way to calculate how many additional individuals would be needed to sample 100% of asymptotic species richness of a sample/region - iNEXT?

I am starting to use SPADE and iNEXT R packages. I think I have finally got my head around most of what I need so far but... Chao et al, (2009) present an Excel calculator to calculate how many...

15 June 2020 1,765 2 View

Statistical test help - moderated regression or 3 way anova?

I am proposing a research study where I am measuring peoples personality (using BFI) across 9 years, as well as the life events they have experienced and their perceived stress. I want to see...

02 June 2020 5,669 4 View

Recently found these contaminants growing in 12 well insert membrane plates, fungal?

These are nasal epithelial cells that have been in culture for several weeks. We have grown 20 different samples of the same material using the same protocol with no such issues. Hoping this is an...

10 February 2020 1,597 2 View

Trouble shooting with plasmid visualization on agarose gel from E.coli ??

Dear Friends, I am trouble shooting since a long for isolation of my cloned construct in the strain E.oli DH-5 alpha. I have tried Qiagen kit but yield is very low to...

23 November 2019 9,475 2 View

Solution to overcome the diffuse band from SDS-PAGE protein gel and can it be used for western blot?

I have run my Crude extract protein from rice shoot sample and as per my observation I feel that my protein is getting diffused as lower part of my running gel. The gel solutions are freshly...

06 July 2018 1,876 3 View

Is there a problem with buffer as I get no signal with 2D-PAGE or Western blot ?

I am trying to measure phosphorylation of lamin A subunits by 2D-PAGE. I resuspend Vero cells in Rehydration buffer (which contains 8M urea) and sonicate them. I run this cell lysate sample on an...

26 October 2015 3,471 2 View

Can I use same tissue homogenised in PBS for different biochemical tests where kit has a different lysis buffer suggested?

Can I use same tissue homogenised in PBS for different biochemical tests where kit has a different lysis buffer suggested?

01 January 2015 5,449 4 View

Sample dilution for quantitative analysis using ELISA?

If the detection range is in ng/ml but the reference range is in ug/ml for a molecule or protein in serum or plasma .how to dilute and what is the initial volume to be taken for quantitative analysis

02 March 2021 7,670 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

IF troubleshooting: looking at an intracellular protein that localizes to the cell's and how to reduce increase in signal due to cell ruffles?

I am currently trying to stain for PI3KBeta in MDA-MB-231 cells. My current issue is that 1) beta stains all over the cell and 2) there is an increase in signal at the edge of the cell and this...

24 February 2021 8,582 1 View

Is it efficient to do liquid biopsy to investigate the gene expressions of the tumor?

To do liquid biopsy and look for specific gene expressions there are so many published papers. Thera are different protocols and procedures used to isolate circulating cell free RNA or mRNA from...

23 February 2021 7,326 5 View

How to clean up TEM grid with plasma cleaner?

I need to clean the hydrocarbon contamination on the carbon substrate of TEM grid. However, I heard that substrate could be broken by the powerful plasma. My substrate is pure carbon, with ~3nm,...

23 February 2021 3,262 5 View

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17 February 2021 4,217 3 View

What are the best cations for preparing competent cells, Ca2+ or Mg2+?

I have been reading various articles and most state that treatment with CaCl2 produces higher efficiency of transformation than MgCl2. Can anyone explain why Ca2+ is better than Mg2+ at...

16 February 2021 8,519 5 View

How can I spin a thick layer of organic polymer?

I need to spin a 120um thick film. My current procedure consists of spinning the polymer (about 20um), uv exposure, plasma etch, and then spinning another 20um thick layer, uv again, plasma etch...

10 February 2021 2,055 6 View

What is the general miRNA yield one gets from the Qiagen miRNEASY serum/plasma advanced kit?

I am using the Qiagen miRNEASY serum/plasma advanced kit, using 400ul of frozen plasma. What is the yield you get on average? I am using nanodrop currently for quantification. I am getting...

02 February 2021 8,806 2 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