Reverse effect of FCCP in respirometry?

HI, We all know by literature by adding FCCP we can uncouple the mitochondrial membrane potential and increase the rate of respiration. When I tried/ titrated with a cell line, instead of increase...

31 December 2018 6,629 1 View

How to define Corrected Total Cell Fluorescence (CTCF) and what is the Units for it?

Hi, Could some one explain me " What is Corrected Total Cell Fluorescence (CTCF) ? " Does CTCF has any units? Thank you

08 September 2018 1,263 0 View

96 well plates for absorbance at 450nm?

Hello friends, Is it okay to use regular tissue culture/cell culture 96 well plates for measuring absorbance at 450nm? Although the wave length is in visible range I am bit skeptical about that...

06 July 2018 6,399 2 View

Primers to confirm guide ligation into Px459?

Hi, I am trying to ligate/insert the phosphorylated/annealed guide sequence into the Bbs1 digested CIP treated px459. I am getting the bacterial colonies on the AMP agar plate. but when I am...

02 March 2018 582 7 View

Generation time Vs doubling time?

How the generation time of bacteria varies from doubling time? Could some one help me understand the concept. Thanks

01 February 2018 7,697 6 View

What are crispr cas stable cell lines?

Hi, I have just started to learn/adapt CRISPR system for our research and I found few companies providing CRIPSR stable cell lines. Unfortunately, none of them explained to the level where a...

11 December 2017 2,503 2 View

Why do we need to incubate plasmid and bacterial cell mixture on ice during Transformation?

02 March 2016 9,317 5 View

Is it necessary to dilute Accutase to dissociate cryo preserved mouse neurospheres ?

I am trying to dissociate primary neurospheres (mouse), which are cryopreserved in Liq.N2. I followed the standard procedure given by the supplier and the same procedure was followed in many...

01 February 2015 1,080 0 View

What is single cell biology

08 September 2014 6,161 5 View

Why should we add FBS to the serum free media?

We all know, during few cell culture experiments we use serum free media, we still add FBS (Fetal Bovine Serum) in some cases. We are also aware that FBS provides the growth supplements for the...

07 August 2013 3,298 30 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

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

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

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 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