Why do cells not attach after trypsinization?

Hi all, Currently, I am having an interesting problem. Although I worked with the cell line, dld-1, before without any issue, this time, they dont attach the flask after trypsinization. Has...

11 December 2019 3,590 3 View

Silenced gene does not affect downstream target genes?

Hi, I silenced a gene (LSD1, KDM1A) using 2 different shRNA in 3 different cell lines. When I check its mRNA level, it is more than 70-80% silenced. However, if i check its downstream target (at...

03 April 2018 5,872 3 View

Calcium Phosphate Transfection Problem?

Hi, I am trying to transfect 293T cells using 2M CaCl2 and 2X HBS (50 mM HEPES, pH 7.07, 10 mM KCl, 12 mM dextrose, 280 mM NaCl, 1.5 mM Na2PO4) However, some plates, still have particles (probably...

02 March 2018 4,689 6 View

Cannot show reduced lncRNA level using siRNAs?

Hi all, I am trying to perform a loss-of-function assay. I silenced some lncRNAs which I found them from a high-throughput screening considering changes in their downstream molecule. In order to...

09 October 2017 3,855 0 View

Alternative to Southern Blot?

08 September 2016 4,045 3 View

Having problem with mutant IDH (Isocitrate dehydrogenase) enzyme overexpressed cells?

Hi, I am having trouble with cells overexpressed mutant IDH enzyme. Problem is that it does not work. In other words, I chose some genes as a positive control from other papers and evaluated their...

02 March 2016 8,254 3 View

No title

08 September 2015 4,775 2 View

How can I protect RWPE-1 cells from any contamination?

06 July 2014 8,632 11 View

Is there any software or website for matching sequences and diseases?

03 April 2014 8,918 4 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

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

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View