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Questions related from Sandeep Kumar
I'm getting zero or no ct value in my gene expression analysis during RT-PCR analysis. I'm not understanding the meaning of zero ct value, how I should interpret the data now. Should I consider...
03 March 2020 3,143 3 View
I'm using transparent polystyrene 96 well plate for H2dcfda assay, is it ok or I should use black plate with transparent base.
02 February 2020 5,344 3 View
I have treated THP1 and AGS cells for 12, 24 and 48 hours with a bacterial toxin concentrations 0, 5, 10, 20, 40, and 80 ug/ml. Now I want to prove my results with statistical methods but I'm...
11 November 2019 6,554 6 View
Hello everyone, I'm digesting 2 ug of midiprep isolated pET15b and 22b with BamHI and XhoI at 37 oC for overnight. There is smearing in gel and size of the cut vectors also not appearing actual,...
06 June 2019 1,654 4 View
I'm analyzing 445 protein sequences of a single protein for phylogenetic analysis. Multiple sequence alignment of these sequences is showing gaps of different sizes. Is it ok to keep these gaps...
04 April 2019 8,569 3 View
I'm preparing tris buffers of molarity 1.5 and 0.5 M with respect to pH 8.5 and 6.5 respectively. The conductivity of molar concentration 1.5 is 26.0 ms/cm and for 0.5 M is 33.6 ms/cm. Is it a...
04 April 2019 6,346 9 View
I have downloaded 1000 protein sequences having very big name in FASTA format. I want to cut short these name in a quick manner. Is it possible through and command line or software or I will have...
04 April 2019 7,387 4 View
I'm getting more than 200 colonies after transforming 2 ul of my ligation mixture from 1;3 ratio. I used 100 ng of vector and now it is going hectic to screen a positive clone because the colies I...
02 February 2019 8,822 4 View
Hello every one, I'm getting long streaky colonies after transforming pET15b in DH5alpha. Kindly suggest the reason.
02 February 2019 6,692 7 View
I'm cloning a secreted Gram negative bacterial toxin and not finding any colonies with DH5alpha and pET15b. I'm just wondering what could be the best competent cell for keeping and expression of...
01 January 2019 9,016 8 View
Are there techniques other than SEM to confirm bacterial outer membrane vesicles. Actually, our SEM is out of order for some time, so, I want to confirm them by some other techniques.
12 December 2018 9,564 4 View
I have gel purified double digested vector and insert having concentration 1 ng/ul and 6 ng/ul respectively. I have tried ligation thrice at room temperature but did not get any colonies. I'm...
10 October 2018 1,640 21 View
I'm digesting pET15b and my insert with NdeI and XhoI which are adjacent to each other in this vector. In insert I have only added three base pair in the 5' end. In sindle digestion, both the...
10 October 2018 1,072 18 View
I'm using 0.7% agarose gel for elution of restriction digested plasmid DNA and my insert ranging from 2.3 kb to 5.7 kb from two different brands having gel strength >1200g/cm2 (1.5% ) and...
10 October 2018 6,098 5 View
I have transformed DH5@ with 2 ul of an old stock of pET15b and pET28a through heat shock method. Competent cell was prepared through method described by Inoue et al. Heat shock was performed for...
09 September 2018 3,532 15 View
I have isolated pET15b and pET28a, run on 1% agarose gel, they are showing the size exactly around 5.7 and 5.4 kb. I'm just wondering when I will digest them what will be their size and which one...
08 August 2018 3,832 4 View
I'm cloning a Gram negative bacterial secreted toxin in pET15b and pET28a vectors. I have confusion that which strain of E. coli would be better for induction of protein expression, BL21 or BL21(DE3).
08 August 2018 2,858 5 View
I did a gradient PCR for 16SrRNA with universal primers 27F and 1492R. The PCR product size is around 400 bp instead of 1.5 kb. What could be the possible reason. the annealing temp. was ranging...
08 August 2018 1,943 0 View
Protein carbonylation blot is not showing low molecular weight proteins and also there are spots in background.
07 July 2018 8,078 3 View
I want to clone a secretory protein from H. pylori and transformation will be done in E. coli BL21. The protein has a 33 aa signal sequence in its N terminal and auto transporter domain at C...
07 July 2018 9,565 2 View
What is the importance of Kozak sequence in protein expression during cloning?
07 July 2018 1,822 4 View
I want to study the evolutionary impact of two proteins on each other from a single species, can it is possible to study through phylogenetic or computational methods and what tests and parameters...
07 July 2018 8,506 2 View
I want to load 50 ug/20 ul/well of SDS-PAGE for Western blot and I have protein concentrations 6.18, 4.9, 5.76, 6.53, 4.43, 5.83, 5.11& 7.46 ug/ul. Suppose I calculate through formula...
06 June 2018 7,898 6 View
I have seeded 10000 cells/100 ul media/well of a 96 well plate and want to treat them with 1, 2, 5, 10, 25, 50 & 100 ug of a protein extract, having concentration 1.8 ug/ul,to know the...
06 June 2018 4,031 5 View
What should be the significant no. of cells we should seed for any cell culture assay. Does the cell no effect outcomes of the results.
06 June 2018 7,294 4 View
I'm just wondering about the technical and biological replicates, what are these and how many no's we should used for a cell culture assay.
06 June 2018 3,388 0 View
I want to see the effect of a bacterial protean in THP1 and AGS cell lines. What would be the exact protocol for serum starvation of these cells.
05 May 2018 805 5 View
I want to perform SDS-PAGE for crude extract of bacterial protein and cell lysate for Western blot analysis. It is in literature that Tricine-SDSPAGE has better resolution than Glycine-SDSPAGE....
05 May 2018 4,404 4 View
We have an ELISA plate reader in our lab having an option path length correction. I'm just wondering when we should use this function and how it works.
05 May 2018 7,706 3 View
I have precipitated crude proteins from extracellular media of a bacteria with up to 80% ammonium sulphate precipitation. I'm performing dialysis to desalt the protein sample in 1X PBS pH 7.5....
05 May 2018 4,391 4 View
Is there any method through which we can perform MTT assay for different time points in a single 96 well plate. I'm treating cells with six different concentration and now want to see there...
05 May 2018 1,536 4 View
I have designed forward and reverse primers between BamHI and XhoI respectively to clone a 1-360 bp segment of bacterial gene pET-30 a (+) (5422 bp). I'm designing a cloning primer just first time...
01 January 2018 4,015 3 View
I'm culturing AGS and THP-1 cell lines. I want to confirm whether these are same cell lines or some one else. What characteristics we can analyse other than normal microscopy.
01 January 2018 6,526 3 View
I'm diluting 1, 2, 4, 8, 16, 32 & 64 ul of 10mg/ml of BSA solution in to 99, 98, 96, 92, 84, 68 & 36 ul of PBS buffer, making total 100 ul. Now adding 50 ul of Bradford reagent. I'm...
01 January 2018 4,759 4 View
I'm not getting color in my Bradford assay, so, just wondering that what should be the protein sample, Bradford reagent and buffer ratio in Bradford assay to obtain appropriate color?
01 January 2018 8,582 0 View
I want to clone a 1290 aa long bacterial secretory protein having signal sequence (33 aa), autotransporter domain (at C terminal) and hydrophobic domain (acts for main function of protein) and...
12 December 2017 952 4 View
Kindly recommend the best books for protein isolation with principles and practice methods
11 November 2017 3,424 0 View
We want to purchase two workstations for plants and microbial metagenomics, molecular dimension (MD) simulations and docking studies of macromolecules. What would be the best specifications and...
11 November 2017 9,691 4 View
I need to degas buffers for column chromatography, how we can perform it in a lab without any sophisticated instruments. We have a small vacuum pump.
11 November 2017 9,703 3 View
I want to isolate a protein through anion exchange chromatography using Q sepharose. The theoretical PI of protein is 9.02, Molecular weight is 139312.18, Number of amino acids is 1290, Total...
11 November 2017 2,190 3 View
I want to study the folding of a protein whose structure is not known yet, through molecular dynamics and simulation, is it a right method to solve this problem.
10 October 2017 7,043 11 View
I want to purify a 88 kDa bacterial secretory protein, which oligomerizes into different forms such as heptamers, octamers, dodecamers etc., for that I have buy Q Sepharose, Sepharose 6B phenyl...
10 October 2017 7,190 1 View
I'm analysis D and L glucose absorbance through UV spectroscopy and finding differences in absorbance for same concentration, what is the reason behind it.
09 September 2017 5,396 6 View
What are soft matter polymer system? Does the protein soltuion come in this catageory? In what way we can study this system with SAXS and SANS.
09 September 2017 7,628 1 View
I want to detect effect of a bacterial toxin in human cell line and want to see cell viablity, so which assay will be better, many people critisize MTT.
09 September 2017 2,876 8 View
Nrf-2 is a modulators of different genes, so, why it is used as a marker for proteasome lysis analysis?
04 April 2017 2,561 1 View
What are plasma free amino acids?
03 March 2017 4,673 1 View
What should be the column height and diameter ratio in protein size exclusion chromatography?
12 December 2016 2,756 9 View
I want to isolate secretory VacA protein from a broth culture. After salting out, I'm performing dialysis with 1X PBS (pH 7.5) and passing 1 ml of sample through self made 2 ml Sepharose 6B column...
12 December 2016 216 7 View
We want to purify a 88 kDa bacterial protein from extracellular crude extract through gel permeation/size exclusion chromatography. I'm just beginner in this field and wonder about the basic...
12 December 2016 7,100 4 View
Suppose if mRNA is showing up regulation of a gene through qRT-PCR, then, is it necessary that the level of corresponding protein will also be high in western blot.
09 September 2016 3,501 5 View
I want to detect the inflammasome formation in THP-1 and RAW macrophages against the treatment of a bacterial toxin. So, is it mandatory to treat these cells with PMA/LPS/Nigercin etc.
09 September 2016 3,740 3 View
We are studying the effect of a toxin on cell membrane lipids which binds to receptors RPTP-alpha & beta but their localization in lipid rafts of cell membrane is not well characterized, so,...
09 September 2016 4,224 2 View
What are the different ways of studying role of SNARE proteins during bacterial toxin induced inflammation. How to study the effect of a bacterial effector in phagosomal fusion.
09 September 2016 1,942 1 View
How to check host cell membrane integrity disrupted by a bacterial toxin.
09 September 2016 6,384 0 View
How to study depolarization of mitochondrial membrane caused by a bacterial toxin.
09 September 2016 7,426 0 View
We want to see expression profile of epithelial cell line infected by a pathogen in the presence and absence of ubiquitins?
08 August 2016 7,585 1 View
Cryo-electron microscopy
04 April 2016 3,147 9 View
no
04 April 2016 2,261 0 View
We want to tag a protein with FITC for its localization studies, what are different ways.
01 January 1970 1,332 0 View
We have a DSC instrument, DSC3500 Sirius from NETZSCN in our Physics lab. They are doing material analysis from it. I want to know whether we can perform protein or DNA analysis in it.
01 January 1970 6,227 3 View
Kindly help me by suggesting the mechanism of different antibiotics and supplements used during culturing of H. pylori.
01 January 1970 4,485 4 View
