Regarding cloning of Nus His tag fusion construct, to express double tagged fusion protein ?

Strategy 1) I have prepared N- Nus and C- His(6-His) tagged fusion construct of my protein of interest in pET43.1a by using SpeI and XhoI restriction enzyme and Prescission protease recognition...

09 October 2017 2,905 3 View

Sonication of protein sample?

For the expression of subtle Nus tag fusion protein I have optimized sonication for 5ml culture, 50 percent amplitude, 1pulse off 1pulse ON for 30 second. With repeated cycles with the gap of...

05 June 2017 8,975 10 View

How to calculate bacterial cells /ml from OD600 in Rosetta cells?

I want to calculate total number of Rosetta cells by using OD 600 , please explain.

04 May 2017 1,598 7 View

How much concentration of Protease inhibitor cocktail should I use in buffer?

How to decide how much concentration of protease inhibitor cocktail is required in buffer for protein expression ?

04 May 2017 6,702 3 View

150 mg/ml Rifampicin preparation in methanol ?

02 March 2017 8,268 5 View

Regarding linear and exponential PCR amplification during quick change and Inverse PCR site directed mutagenesis (SDM)?

10 November 2016 5,582 2 View

How to select molar ratio for cloning ?

I am interested to know whether a 1:3 molar ratio is ok with a 5.4 kb vector [5.4 kb after double digestion,(actual size of vector is 7.2 kb) ] and 600 bp insert DNA ? Is there any relation for...

09 October 2016 2,707 5 View

Regarding polynucleotide kinase (PNK) phosphorylation and heat inactivation after ligation of site directed mutated DNA ?

I am using NEB mutagenesis protocol (not kit) for point mutation, primers are designed back to back orientation and after PCR I want to perform PNK phosphorylation. I have thermoscientific PNK...

09 October 2016 1,545 1 View

Interpretation of gel before and after DpnI digestion?

09 October 2016 6,722 6 View

Not getting desired length of PCR product with Q5 polymerase ?

I amplified human gene cDNA(600 bp) with Q5 pol but after amplification I got 500 bp band with some nonspecific bands of less than 500 bp, then I performed gradient PCR and again I got 500 bp band...

09 October 2016 8,405 7 View

Looking for Materials with high thermal expansion coefficient and high temperature resistance?

03 March 2021 8,272 1 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

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 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 calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 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