Extra DNA band in DpnI based point mutation?

There's a clone in pBAD18cm vector (6800bp) which was subjected to point mutation through PCR-based technique. After PCR and DpnI digestion an extra band of approx 1500bp was observed in cloned...

01 February 2016 4,561 4 View

What are the best free softwares to study and analyse enzyme kinetics data?

I am doing enzyme kinetics studies. I need to derive certain parameters for a representable data. The present MS excel sheet fails to provide enzyme kinetics parameters such as Km, Kcat, Vmax...

10 November 2015 7,277 7 View

What is the best solvent for carbapenem antiobiotics? Water or DMSO?

A very basic query regarding the solubility, stability and storage of carbapenems antibiotics such as Meropenem trihydrate or Imipenem monohydrate. I have known to dissolve them in DMSO in higher...

09 October 2015 5,403 8 View

Are there any ghost bands in my SDS-PAGE gel or contaminating proteins?

Here I have attached SDS-PAGE gel picture of Ni-NTA affinity chromatography elution profile of a protein at varying concentration of imidazole in tris buffer(pH 8). I have done washing in 10, 25...

07 August 2015 1,667 8 View

How do I get higher molecular weight band after PCR clean up followed by gel extraction of double digested inserts?

It's confusing to find extra bands of higher molecular weight in my insert sample which was PCR purified by gel extraction method. The insert bands were very clean in 0.8% agarose gel. It was...

05 June 2015 4,240 13 View

Is it possible to get a protein of interest at higher molecular weight than expected in SDS-PAGE?

I am checking in-vivo expression of a protein(full length) in pBAD18cm vector using BL21 expression host. I am getting an extra band of protein, 10kDa higher than expected in SDS-PAGE as compared...

04 May 2015 6,479 10 View

What are the good servers for detecting signal peptides apart from SignalP 4.1 and Signal blast?

What are the good servers for detecting signal peptides apart from SignalP 4.1 and Signal blast? Sometimes these servers fail to detect the presence of signal peptide in a given protein sequence....

04 May 2015 8,435 2 View

Does NDM-1 have signal sequence?

I tried to check the presence of signal sequence of full length construct (amino acid) of NDM-1 using SignalP 4.1 server but could not find it. Can any body suggest anything in this matter? Thanks

04 May 2015 8,397 0 View

Why there are two bands in SDS-PAGE of a purified protein instead of one?

I have cloned a beta-lactamase gene in BL21 E.coli strain using pET28a vector for expression and in vitro studies. After Ni-NTA column purification of this His Tagged protein, I am getting two...

31 December 2014 2,330 16 View

What should be the ideal dialysis buffer composition for a His-tagged purified protein to remove imidazole salts?

My His-tagged purified protein contains 10mM trisCl, 300mM NaCl and 200mM imidazole at which the protein was eluted at pH 8.0. What should be ideal dialysis buffer composition to remove...

31 December 2014 5,867 10 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

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02 March 2021 3,060 3 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View