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Questions related from Nasir Javaid
Hi, I extracted total cellular RNA from HEK293T cells by using a commercial kit. I left samples untreated or treated with 2ul RNaseA/T1 mixture (invitrogen) in the presence of PBS or protein...
12 January 2024 2,416 4 View
I am getting smear on gel after using linear DNA (amplicon) as a template while this is not the case with circular DNA (plasmid) as a template with same set of primers. I have used various amounts...
07 July 2023 7,280 8 View
I extracted genomic DNA from two different cell types (HEK293T and HDFs) and performed PCR reaction by using a same primer set. The PCR is positive with HEK293T template but not with that from...
07 October 2021 9,412 5 View
Dear all, I bought a recombinant human MCP-1 protein from Thermo FIscher and trying to optimize the signaling activation by using target cell X. I followed the company's instruction to dissolve...
01 June 2020 4,834 0 View
Dear All, I got 2 weeks old paraffin blocks of tumor masses. I want to extract total RNA from them. The available reagents in my lab are: 1) Cell dissociation buffer (GIBCO#13151-014) 2) TRI...
31 March 2020 5,290 5 View
Before buying the reagents specific for particular germ-line, I want to get a clue about the differentiation potential of my stem cells (iPSCs) in vitro. For that, what are the minimum essential...
30 May 2019 2,716 3 View
Previously, I successfully knocked-in a transgene into HEK293 cells by using paired nickase CRISPR system. Now, I am trying to do it with human primary fibroblasts (nHDF). I co-transfected 3...
16 October 2018 508 4 View
I am using the TeSR-E8 medium for the maintenance and growth of iPSCs. I made the aliquotes of complete medium without adding Pen/Strp and Normocine, and stored at -20C. On need, I thaw one of...
07 October 2018 7,493 10 View
I am trying to validate TNF-alpha inhibitors using nHDF (Human Dermal Fibroblast). According to the literature protocol, ActinomycinD (ActD) is applied to block the transcription followed by...
05 October 2018 7,662 2 View
I am using a compound X (TNF-alpha inhibitor) which turned out to be highly toxic at 10uM and 20uM. In case of IL-8, I can see the dose dependent reduction from 1uM to 10uM to 20uM (mirror of cell...
02 October 2018 3,137 8 View
My cells are transfected with GFP plasmid. I want to detect a particular cell surface marker by immunostaining. Can I use Alexa Fluor 488 dye containing secondary antibody? Will it overlap with...
28 September 2018 4,652 6 View
I found an un-opened bottle of expired (2015) KO-SR (serum replacement) for iPSCs at -20C in my lab. The catalog number is 10828028 from GIBCO. We are facing some funding issues so it is hard to...
24 August 2018 2,998 2 View
I did ROS assay using H2-DCFDA but could not find the function of detecting 2 wavelengths ( ex: 485, em: 520) at the same time as single reading in microplate reader so I got readings separately...
02 July 2018 6,745 5 View
I did LDH cytotoxicity assay on MKN-1 cell line with my compounds at various concentrations. After 24 hours of incubation, LDH assay was performed according to manufacturer's protocol. There is...
02 July 2018 4,640 7 View
It seems to be easy process but I can hardly cut them separately under UV light (long exposure to our body is hazardous). I used 1% agarose gel so far and ran for 2 hrs at 50V. If someone has...
19 June 2018 280 4 View
After digesting vector with AsiSI and NotI, I gel purified and quantified. The insert was PCR amplified, gel purified and cut with same enzymes. After cutting insert, I did not gel purify but heat...
05 June 2018 8,860 7 View
If we want to check the effect of a small molecule on various cell lines of different sizes then which one we should keep same among them so that same amount of molecule will go in each cell among...
24 May 2018 9,102 4 View
Dear Researchers! I am facing a serious issue regarding making stable HEK293T cell line using CRISPR/Cas9. I am using paired nickase Cas9 along with cut and uncut Donor plasmid (separately). After...
05 May 2018 5,734 3 View
Can we activate the cytokine secretion in THP-1 monocytes by using the flagellin? Do we first need to differentiate monocytes into macrophages before applying flagellin? I came across one...
28 March 2018 2,043 3 View
I tried to integrate transgene into mammalian cell line using CRISPR technology. After 48 hours of transfection, I isolated genomic DNA using SP-DNA tissue kit (SP-DT)...
24 March 2018 1,382 1 View
Can anyone please recommend the best tool to predict off-targets? How many minimum mismatches and RNA/DNA bulges must be considered with reference to PAM sequence? I am using the spCas9 in my...
23 March 2018 1,455 2 View
I thawed a 10 years old stock of J774A.1 from liq. N2 and tried to grow in 6cm dish using High glucose DMEM supplemented with 10%FBS, Pen/Str and Normocine. It's been 16 hours to culture but cells...
20 March 2018 9,910 4 View
I am trying to reprogram MEF and HDF into iPSCs using trasfection reagent like X-trem Gene, Lipofactamine2000 and Lipofactamine3000 but the transfection efficiency is low. I also have NEON...
02 March 2018 329 3 View
I am trying to generate iPSCs by retransfecting my target cells (HEK293T) grown on feeder cells. I grew Mit-C treated feeder cells (70% confluent) on Gelatin-coated plate followed by seeding...
21 February 2018 9,127 7 View
I am trying to generate stable cell line (HEK293T) by using the X- plasmid. I got negative result by using the intact (circular) plasmid so this time I am trying to use linearized one. The problem...
10 January 2018 3,257 5 View
I am trying to transfect a plasmid in HEK293T and nHDF cell lines by using Neon electroporation system and have following 2 questions: 1) After successful transfection, what is the expected time...
28 December 2017 8,039 3 View
I have two plasmids: plasmid A (8434bp)and plasmid B (12,082bp). I want to do co-transfection of these plasmids in different ratios of molarity by keeping the total DNA amount same (1.5ug). The...
25 December 2017 8,950 4 View
Hello everyone, I am trying to construct a plasmid vector with two promoters in opposite direction (one in clockwise and other in anticlockwise direction on circular map). Should I add spacer...
29 July 2017 8,153 3 View
Hello everyone, I am trying to make iPSCs by using Yamanaka factors (OSKM). I am confused that should I use human derived factors or mouse derived? At this moment, my target is to use human cells...
29 July 2017 5,042 4 View
Hello everyone, I am thinking to use mPGK promoter for expressing my gene of interest in human cell lines (first) and later in mouse cell lines. Will it work in human cell line in a similar way...
29 July 2017 3,688 2 View
Hello, Firstly, Can someone provide me sequence of Tet-on Promoter and Blasticidin marker gene? Or can you recommend me reliable source from where I can easily download it? Secondly, I isolated...
16 May 2017 9,358 2 View
Hello, I am trying to confirm ligation (after cloning) via colony PCR using GoTaq Green Master Mix. Tm of FP and RP is 50C and 58C, respectively. Applied annealing temp so far 44C, 48C, 50C, 52C,...
25 April 2017 8,533 3 View
