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

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it? Thank you in advance

03 March 2021 3,568 1 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

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Genome-wide gene identification protocol ?

Is there any book chapter/book, webpage or research article available to understand genome-wide gene identification?

28 February 2021 8,095 1 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

Errors in DNA sequences?

I am trying to better understand the scope of DNA replication and sequencing errors, e.g. 1. I have seen similar error rates of 10e4 to 10e5 for cell & instrumental DNA replication,...

24 February 2021 4,397 3 View