How did such a large plasmid isolated and sequenced?

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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 4,691 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 7,413 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 5,545 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 1,836 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 4,415 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 6,670 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 8,562 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 8,562 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 2,479 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 6,007 10 View

Why Do TDS and EC Increase with Larger Wastewater Volumes, While BOD and COD Decrease?

I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...

09 August 2024 9,621 0 View

Could it be a cell culture contamination?

Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...

09 August 2024 2,824 3 View

How to confirm the site-directed mutagenesis result without performing NGS?

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...

08 August 2024 4,645 2 View

I can't see the ssDNA band after performing asymmetric PCR. Is there any way to do this?

After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...

08 August 2024 1,668 3 View

Is there any way to quantify bacterial and fungal cells in their mixed culture?

I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...

07 August 2024 7,535 4 View

How to normalize and take the significance of the MTT OD values with 3 replicates for the same cell-line?

Hi, I have a question about normalizing the MTT OD values for doing the statistical analysis. So, if we have 3 different plates and we call them 3 different replicates, so, first we would...

07 August 2024 8,106 4 View

Why activated CAR-Jurkat cell could not kill targets?

Previously when I co-coluture anti-CD19(FMC63) CAR-Jurkat with Raji with E:T=5:1, Jurkat can eliminate Raji in 24h. However, when I test another CAR construct, although I can dectect totally CD69...

06 August 2024 641 2 View

How much total RNA concentration to be extracted from sorted plasma cells from bone marrow of C57BL/6 mice for RT-PCR ?

i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...

05 August 2024 8,835 1 View

Where should the ARS sequence be introduced on a plasmid?

Hi all, I need to introduce an ARS (autonomously replicating sequence) in my plasmid but I'm not sure which position would be the best. Does anyone have any suggestion? A picture of the plasmid...

05 August 2024 1,573 4 View

Why after performing site directed mutagenesis ,I don't see any colony after transformation?

I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...

05 August 2024 9,059 3 View