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

SiRNA Transfection to COLO205, SW620, HT29?

Hi, Is there anyone who has done siRNA Transfection in COLO205, SW620, HT29 especially using either RNAiMAX or Lipofectamine3000? Besides do you have any experience to transfect/transduce plasmid...

11 December 2017 1,759 4 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

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

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

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View

What urea buffer can i use for biotagged protein extraction from streptavidin beads?

Hi all, I have been doing research on biotagged LDB1 proteins in Mel cells. I purify the proteins using M280 streptavidin beads. The yield is very low so I've been trying to optimize it by...

24 February 2021 5,749 3 View

Can I use a protein-devoid condition as my negative control in ELISA?

Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...

23 February 2021 7,429 1 View

Is there a way to extract total RNA and protein from cell culture at the same time, without denaturing protein?

Hi all, Is there a way to extract RNA and protein from the same cell culture sample? Because we need to use the protein for gelatin zymography at later time, we are looking for a method that can...

22 February 2021 5,341 5 View