Has anyone had issues staining AAV injected tissue with immunofluorescence?

I am injecting the hippocampus with an AAV8-GLT1 viral vector. I noticed when I am staining with immunofluorescence the secondary antibodies (Ms 598) is sticking to AAV virally injected tissue...

19 March 2020 5,961 5 View

Can anybody help me with the protocol on how to detect protease activity in a gel containing Casein substrate?

I have just cloned and got a soluble expression(50%) of a thermophilic serine protease and did the heat treatment for purification at 70 and 80 degree with over 90% purity, and also have a...

15 March 2019 9,330 3 View

What are best purification buffers and their conc. that I can use to purify a 6xHis- tagged recombinant serine protease using Ni-NTA resin ?

I have just cloned and expressed a thermophilic alkaline serine protease in E.coli BL21(D3). The protease was expressed as a soluble protein (about 60%) with about 30kDa Mol. wt. (confirmed by...

27 December 2018 1,777 7 View

Can i cytospin cells that are already fixed and stained for IF onto glass slides for confocal imaging?

I plan to isolate breast cancer cells from patient blood samples and would like to fix and stain them in-situ, within the Eppendorf, and then spin them onto a (poly-l-lysine-coated) glass slide...

14 August 2018 3,354 3 View

Can MOLT-4 cell be stimulated with anti-CD3/anti-CD28?

I am looking for a cell line other than Jurkat cells that can signal through the TCR. Would this be possible with MOLT-4 cells or do you know of other cells lines that I could use?

07 June 2018 651 3 View

I'm running a comeptitive ELISA and when i add my 0.15M HF stop solution (with 0.1MEDTA) the net OD decreases and it should increase. help!?

I'm running a comeptitive ELISA and when i add my 0.15M HF stop solution (with 0.1MEDTA) the net OD decreases and it should increase. help!?

31 May 2018 9,776 2 View

What is best temperature for the incubation of thermophilic bacteria on liquid broth for the purpose of DNA extraction?

I incubated my organism (thermophillic bacteria) on LB broth at 37 and 40 degrees centigrade for the purpose of extracting DNA from the organism but did not show any growth after 22hrs of...

05 October 2017 601 11 View

Are there any publically available DNA database for recA gene sequences to use in metagenomic analysis?

14 August 2017 1,969 2 View

Collaborative research on investigating changes of cannabis flower throughout curing?

21 May 2017 4,620 2 View

Is anyone aware of smart phone apps or programs to measure the raw acceleration signal from the phone?

Looking for a way to measure, record, and export raw acceleration from the smart phone's internal accelerometer.

30 November 2016 7,438 6 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 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

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

Difficulty in cloning of 1kb gene into vector?

I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting...

25 February 2021 3,221 7 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