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Questions related from Nasir Javaid
I am trying to make iPSCs from Keratinocytes (HaCaT). Being a beginner, it's hard for me to judge morphologically about the right clone under microscope. Can you please guide me by looking at the...
02 February 2019 8,929 5 View
Hello, I want to confirm the specificity of my protein to human TLR5. For that, I am interested to buy the invivogen cell line but I came across two types of hTLR5 cell lines: 293-hTLR5 and...
12 December 2018 8,052 0 View
Hey! I used mouse fibrosarcoma cell line (L929) and treated with 1ng/ml rmTNF-alpha and extracted protein at different time points (0min, 15min, 30min, 1hr, 2hr, 5hr, 8hr, 12hr). Then I performed...
12 December 2018 9,179 3 View
I found an old ALP kit in my lab (once bought by seniors) with no protocol inside kit box (may be lost). I could not find the protocol on Sigma website. If someone has the protocol then can you...
09 September 2018 7,628 0 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...
09 September 2018 7,438 7 View
I converted differentiated cells into round shaped de-differentiated cells in F12/DMEM medium. As soon as, I transferred them to maintenance medium (TESR-E8 medium) they started changing their...
08 August 2018 2,002 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...
07 July 2018 8,940 6 View
I am treating my cells (MEF) with three different concentrations (1um, 10um and 20um) of various small molecules (M1, M2,.....,M10). Some of them show dose-independent behavior on cells but others...
07 July 2018 5,194 5 View
On the way to iPSCs. The targeted cells started to become circular (indication of dedifferentiation). Before I go for their confirmation as iPSCs they started to detach from the plate surface....
07 July 2018 4,835 1 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...
06 June 2018 4,967 8 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...
06 June 2018 2,873 4 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)...
03 March 2018 7,637 1 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...
03 March 2018 7,733 3 View
I am trying to reprogram MEF (Mouse Embryonic Fibroblasts) into iPSCs. During the process, cells are shrinking, clumping, dying and detaching from the surface (non-coated) as you can see in two...
03 March 2018 236 9 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...
02 February 2018 9,848 8 View
I am trying to amplify the 10kb fragment using human genomic DNA as template by using the Promega green master mix (M7122) but no result. I am doubtful that polymerase used in this kit might not...
02 February 2018 2,882 4 View
I am trying to make stable cell line (HEK293T) using CRISPR/Cas9 technology. I co-trasnfected CRISPR plasmid along with donor plasmid (carrying Tet-on inducible GOI and GFP + constitutively...
01 January 2018 1,004 6 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...
01 January 2018 2,467 6 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...
12 December 2017 3,835 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...
12 December 2017 6,254 4 View
I am trying to transfect nHDF (normal Human Dermal Fibroblast) cell line with lipofactamine 2000 reagent ( Catalog # 11668019 ) by using 3ul lipofactamine for 1.5 ug DNA (per well of 12-well...
12 December 2017 2,761 2 View
I am trying to knockin a DNA fragment (having Blasticidin selction marker) into the genome of nHDF (normal Human Dermal Fibroblast) and MEF (Mouse Embryonic Fibroblast) cell lines by using...
11 November 2017 2,692 2 View
Hi, I am using online IDT Codon optimization tool (http://www.idtdna.com/CodonOpt) for Pichia pastoris but every time it changes the sequence of optimized DNA for the same input sequence which is...
08 August 2017 8,369 4 View
Hi everyone! I am having problem in growing Huh7.5 cell line. I am using cDMEM (invitrogen 11965) supplemented with non-essential amino acids (1X) and pen/strep. At beginning after thawing and...
04 April 2015 10,006 2 View
