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I ordered two Predesigned assays for quantification of CpG methylation by Pyrosequencing from Qiagen and I do not have any product after PCR. I have dissolved them in TE ph8, tried different...

22 September 2020 3,328 6 View

What does it mean to see a band for uncloned vector and one band for vector with insert from one colony after cloning?

I used gateway cloning to clone my insert into the donor vector. After having colonies, I did a mini prep and ran it on the gel. On the gel I could see a clear band on the right length for my...

14 June 2020 9,829 3 View

What are the soil nutrients use efficiencies in Central America?

Hello everyone, I am working in Central America and I would like to create a document that could help me to calculate "simply" the nutrients disponibility in the soil of coffee fields. The...

10 June 2020 9,671 4 View

How to perform gradient qPCR with Roche Light Cycler 480, to determine the best annealing temperature of my primers??

I would like to determine the best annealing temperature for my primers, giving the lowest Cq and the best efficiency. Is there a thermal gradient feature on the Roche LC480 instrument? And if so,...

16 February 2020 9,471 3 View

Which statistical analysis should I use for 2 dependent variables that are measured with a Likert Scale?

I am studying the generational changes in the relationship between empathy and social media usage. I am using a Likert Scale to measure both empathy levels and social media usage; however, I do...

20 January 2020 9,617 4 View

Is there a way to statistically separate the top percent of performers in a pre-test condition, to then analyse their scores in a post-test measure?

Essentially in a study where there is a pre-test, then an intervention and a post-test (Identical tests), I have a question: "Do students who perform better on the skill test before the...

12 December 2019 9,096 4 View

Why are my re-probings for Western blot analysis unsuccessful?

Hello everyone, I am having problems with re-probing membranes for Western blot analysis. Almost every time I want to re-probe with either the same or a new primary antibody, I don't get any...

11 December 2019 5,066 5 View

How to dissolve and inject 4-hydroxytamoxifen to nude mouse?

What is the normal way to inject 500 ug 4-hydroxytamoxifen per mouse? Is it dissolved 4-OHT in ethanol and diluted with peanut oil at least 10 times?

10 May 2019 3,301 1 View

Why does this strange situation occur in my nude mice xenograft after drug injection?

I injected cancer cells to mice as xenografts. After 7 days, I injected tamoxifen together with an inhibitor of interleukin to nude mice subcutaneously. But 2 days later, the tumor site turned to...

21 March 2019 6,082 4 View

Does anyone have experience in cleaving Fibronectin using an MMP before Western Blot analysis?

We are trying to demonstrate the our treatment effects increases the activity of MMP-2 by showing mutiple cleaved fibronectin bands in a western blot? For this we are going to have a control of...

13 February 2019 7,734 3 View

How to manage western blot for proteomics data validation?

Hi Everyone, I am a bigginer in western blotting and I am trying to use it for validation of my proteomics data. I have: two regulated proteins with molecular mass of 40 and 73 kDa one control...

23 February 2021 6,671 4 View

I can detect my protein but the encoding DNA sequence has stop codons within. Should I ignore the stop codons?

I constructed a synthetic DNA library (scFv, VH-VL orientation) with a 3' reporter and 8x histag. I cloned and expressed this gene following which I performed an ELISA. The ELISA results suggested...

19 February 2021 7,006 5 View

(western blot) can methanol damage my antibody signal?

I accidentally rinsed with methanol after adding my primary antibody to my membrane. I noticed after ~10 seconds and immediately replaced it with PBST (3x15 min washes). Will my antibody signal be...

12 February 2021 5,824 3 View

What is the reason, we got both test and control band on LFA in negative sample?

I am working on Lateral flow assay for virus detection by carbon conjugated polyclonal antibody. We got a high intense band in both test and control line on LFA in negative sample used as buffer....

11 February 2021 6,558 2 View

Background and spots in Western Blot membrane?

Do you know why my membrane looks like this? I did this western for Anti-hnRNP D/AUF1 antibody ab50692, as primary antibody in 1:1000 overnight at 4c and Anti-rabbit IgG HRP linked antibody...

07 February 2021 5,591 4 View

During an anti-His capture ELISA has anyone had issues with histidine residues from secondary antibodies non-specifically binding?

Hi, Hope you can help. Currently been running some sandwich ELISA assays where I coat with anti-His, block and then capture His-tag protein and then sera and then anti-bovine monoclonal...

07 February 2021 7,101 1 View

How do I keep my prefixed (PFA) frozen tissue sections from lifting off the slide?

I'm trying to stain mouse lung tissue IHC-IF I fix in 4% PFA overnight immediately upon harvesting tissues Then dehydrate/cryoprotect in sucrose gradients 15% to 30% Then freeze in OCT media...

06 February 2021 2,336 3 View

Can anyone suggest me broad host range fluorescence expression vectors for Gram-positive bacteria ?

Hi everyone, I am looking for fluorescence expression vectors for expression in gram-positive bacteria present in the skin microbiome. I need to tag them with a fluorescence marker and observe...

06 February 2021 2,566 4 View

Antibody dilution for IHQ in OCT frozen tissue is more similar to IHC in Paraffin-embedded samples or to Immunocytochemistry?

31 January 2021 8,678 3 View

Can i re-incubate a WB membrane with a higher solution of the previously used 1ry ab?

I've incubated my western-blot membrane with both primary and secondary antibodies and i just realized at the end that i've used a lower concentration of my primary antibody for my loading control...

27 January 2021 1,034 3 View