I want to prepare an overexpression module for my protein with a N-ter HA tag. Can you suggest me the possible strategies?

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Can anyone explain me how to design tet-on and tet-off system in mouse and human cell line?

Is there a different methodology in mouse and human expression. I need to check the expression of a gene under the influence of two proteins. Thanking you.

31 December 2018 3,480 1 View

Why do the mutants aren't expressed in the 293 cells whereas Full-length WT construct is expressed ?

I prepared the recombinant protein X-FLAG tagged and two of its mutant. I have checked the mutant was correctly assembled in the pShuttle vector but cannot discover its expression even after 1...

11 December 2018 3,212 17 View

Overexpression construct: I have 6.5kbp size protein for overexpression tool in 293 cells?

I tried twice with HA-tagging. The construct is made using pShuttle vector but failed in overexpressing the construct.

04 May 2018 6,190 5 View

What is the percentage of mutation rate in a cloning of a recombinant protein? How do you proceed for BL21, once the DH5a positive clone is achived?

Please suggest your opinion!

01 February 2018 3,168 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

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Research on Supply Chain Management in Indian context?

Hi, I am planning to apply for the PhD degree in the Supply Chain Mgt. with specific area of "Cold Storage warehouses" during Pandemics and wars. Where lock downs and shut downs are frequent....

02 March 2021 285 2 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 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

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Uroos Akber

I have had the same kind of problem, cloning of the deletion mutants was successful but few of them were not expressed well. I used pcDNA3.0 vector. Then I transferred the inserts into pEBB vector and luckily got the expression. It worked in my case so I shared.

Pratibha Srivastava

Dear Uros,

Thanks for sharing your experience. After repeating it again. I would like to follow up your given method. Thanks!