17 Questions 21 Answers 0 Followers
Questions related from Mansi Srivastava
I have to separate small cDNA fragments (160-180 bp) on agarose gel that will subsequently be used for sequencing. Any recommendation for which buffer is appropriate for running the gel- TAE or...
17 May 2021 6,281 2 View
I am looking for suggestions to improve the yield of my libraries prepared using Illumina True seq small RNA library prep kit. Input RNA is purified small RNA (50 ng) and I run for 15 PCR cycles....
04 February 2020 2,829 2 View
I used Mag-bind total pure NGS beads in 1.2x concentration to clean up my cDNA libraries that are to be used for sequencing. I found that I lost 80% of library during cleanup, is that expected or...
18 July 2019 10,010 1 View
I am trying to check the size of my single stranded RNA on agarose gel with a denaturing loading dye in TAE buffer. Although ssRNA ladder bands are appearing but my samples (having 25-100 base RNA...
11 April 2019 4,234 3 View
It seems to be a broad question, but I am trying to detect NOS2 protein after LPS stimulation. I used following conditions but unable to get NOS2 bands. Please suggest if I need to change my...
14 March 2016 1,453 3 View
Please suggest to me an appropriate protocol for performing NO assay using Greiss reagent. I do not find difference in absorbance reading of control samples and test samples. What can be the...
25 January 2016 1,489 12 View
What should be the concentration of RNA used for cDNA synthesis considering the analysis of low expressing genes in the template. Should the concentration be maximum 1ug or can exceed beyond that?
10 January 2016 934 7 View
Please suggest the optimum concentration of cDNA for RT PCR run. Does diluting the cDNA 1:10 or 1:100 help in increasing amplification, specially for low expressing target genes?
07 January 2016 6,276 4 View
Can anyone suggest me whether Ct values above 35 in RT PCR run is acceptable or not? And whether it signifies primer dimer/ non-specific amplification. What is the acceptable range of CT values in...
06 January 2016 7,220 27 View
I am trying to amplify cytokine genes in cDNA derived from macrophages but unable to get any amplification using comparative delta Ct experiment. Cross checked mRNA concentration and purity which...
06 January 2016 9,846 5 View
I have prepared RNA samples that seem to have protein contamination in them as the A260/280 ratio for them is between 1.5-1.7. Can I use proteinase in the sample and is there a way to get rid of...
28 July 2015 7,963 14 View
I am performing real time to quantify relative gene expression in treated versus untreated samples. How do I plot the ct values in form of gene expression as compared to expression of a...
16 July 2015 6,503 9 View
I am trying to check gene expression through real time but somehow not getting Ct values, in that case what can be the possibilities to troubleshoot this. Can we check whether the cDNA template...
15 July 2015 817 5 View
I am trying dual staining on MCF7 cells but the problem is that the drug used for treatment is somehow getting stuck on the cells/wells and not removed even after washing with PBS 2-3...
10 July 2015 7,236 4 View
Is it added after removing media from each well, and then adding the lipo and DNA mixture dropwise to the well, incubating for 6 hours and then supplementing the cells with complete media (10%...
09 July 2015 6,388 19 View
The compound used for MTT is not soluble in water and sparingly soluble in 5% DMSO, due to which there is issue with uptake in the cells. How can this be solved?
09 July 2015 8,689 14 View
I am performing transfection on MCF7 cells using lipofectamine 3000 (invitrogen) but unable to get any transfectants, instead the cells are dying after incubation with reagent and DNA.
09 July 2015 7,025 20 View
