21 Questions 26 Answers 0 Followers
Questions related from Sushanth Kumar
Hi all, I have been struggling to linearize an 8.4 kb vector by PCR for subsequent Takara Infusion cloning. I am currently using Phusion polymerase with the following protocol 98 C - 30 sec 98 C...
12 December 2019 6,136 4 View
Hi guys, I am over-expressing the protein MLKL along with various tagged forms in an MLKL KO cell line. Despite being able to being able to visualize the protein by both immunofluorescence...
01 January 2019 2,741 6 View
Hi all, I have made an NLS-superfolding GFP (sfGFP) driven in pcdna3.0 and am trying to express it in L929 cells - mouse fibroblast line. Unfortunately, I am not having any luck. The construct...
05 May 2018 1,824 4 View
Hi all, I am attempting to extract AB-42 from some of my 10 month old J20 mice for an Abeta ELISA. I initially perfuse the mouse with heparinzed PBS then snap-freeze one hemisphere on dry ice....
02 February 2018 595 1 View
Hi all, I am having some issues with my Trizol-based RNA isolations. I am attempting to harvest RNA from bone marrow derived macrophages (3x10^6). After the initial separation with chloroform I...
08 August 2017 9,567 11 View
Hi all, I am using the cyclic AMP competitive ELISA kit from Thermo fisher and have been not getting a signal from either my standards or samples. I observe the green color change after adding the...
08 August 2017 5,286 2 View
Hi all, I am performing qPCR on Trizol extracted RNA from BMDMs stimulated with LPS. I am probing for TNF-alpha cytokine production as well as a few other candidates. My TNF qPCR results yielded...
08 August 2017 4,938 4 View
I have a cytosolic protein that I would like to have traffic to the plasma membrane. The protein should not tether to the membrane but be localized to it. I am working with HEK293 cells. 1. There...
04 April 2017 1,311 4 View
Hello, I am using TriLink's Mutagenesis dNTP mix to perform EPR on a 1.3 kb fragment. I have been unable to observe a PCR product when following their recommended cycling parameters. I am able to...
01 January 2017 425 3 View
I have generated an mCherry fusion protein construct in pCDNA3.0. The construct expresses in HEK293T cells as verifed by mCherry fluorescence and immunoblotting against mCherry. When I run my...
11 November 2016 5,045 13 View
I have noticed in several constructs that people will include a triple polyA cassette (typically SV40). Is there a significant improvement in message stability with 3 poly A sequences as opposed...
10 October 2016 7,353 1 View
I am performing a study in which I am observing the interaction between two proteins following a stimulation. Each protein is tagged with half of a GFP moiety and therefore upon interaction would...
10 October 2016 7,661 1 View
I have a 4.8 kb insert that I have been attempting to subclone into pcdna3.0 for months now. In pUC57 the insert seems to propagate just fine and maxipreps give good yields. The following day...
08 August 2016 8,317 4 View
I have a 4.8 kb insert that I attempted to ligate into pCDNA3.1 (~5.4 kb). Transformed ligated plasmid into DH5alpha E. coli. Insert is flanked by HindIII and ApaI cut sites. I digested the...
08 August 2016 7,817 3 View
I am ligating an insert with a repetitive sequence and have been unsuccessful with my ligations using T4 ligase. I have a feeling that my insert is forming secondary structures that are preventing...
08 August 2016 6,754 4 View
I have a fairly large protein (46 kda) that I would like to attach to one of the intracellular loops of a GPCR I am studying. I fear that cloning in the protein in the middle of the GPCR will...
08 August 2016 6,136 4 View
Hi, I am transiently transfecting my CHO cells using the Lipofectamine-2000 protocol. I will then be subjecting my cells to a stress stimulus (mediated through the protein expressed on my plasmid)...
08 August 2016 9,661 1 View
I have a 1.4 kb insert flanked by Kpn-EcoR1 that I am attempting to ligate into a pCDNA backbone. Up to this point, I have gotten no positive clones. I have done successful transformations with...
07 July 2016 5,455 6 View
I would like to subclone in a 25 amino acid group into various intracellular domains of a GPCR that I am studying. I will be making about 8-10 constructs and I will screen for functionality using...
07 July 2016 2,120 3 View
Hey all, I am attempting to transiently transfect three plasmids into CHO cells. My plasmids are a CRE-luciferase (minimal promoter), pRL-Tk-Renilla, and my gene of interest driven by CMV. On my...
06 June 2016 7,874 2 View
I am carrying out transfection of HEK293 cells using Lipofectamine 2000. In a 96 well plate I am co-transfecting a pGL4 CRE luciferase vector along with my pRL-Tk Renilla control vector (50 ng...
05 May 2016 8,584 5 View
