05 May 2016 4 3K Report

I know the protocol for DH5 alfa and BL 21 DE3 competent cell preparation. I wish to know is there any special step or any kind of change in the preparation of Rosett competent cell ?

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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 3,032 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 9,262 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 1,771 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 6,869 3 View

150 mg/ml Rifampicin preparation in methanol ?

I prepared rifampicin (150 mg/ml stock) in methanol  and stored at 4 C but after some day methanol gets evaporated so it makes difficult to pipette out rifampin for next use. If i add more...

02 March 2017 8,396 5 View

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

During Quick change SDM F and R primers are complementary of each other when performing independent Forward and reverse primer PCR reaction  (In different tubes) for  quick change SDM then it is...

10 November 2016 5,755 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 2,838 5 View

Interpretation of gel before and after DpnI digestion?

I have performed Quick-change site directed mutagenesis by using stratagene protocol (Not kit). Mutagenesis primers were exact complementary and  I have performed 1.Independent  F & R...

09 October 2016 6,929 6 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 1,683 1 View

Is PAGE or FPLC purified primer necessary for quick change site directed mutagenesis?

I performed quick change site directed mutagenesis but I am not getting mutated DNA my primers are exact complementary with each other and purification grade is desalted, not PAGE/ FPLC purified....

09 October 2016 8,008 10 View

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Why activated CAR-Jurkat cell could not kill targets?

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Are there instances where molecules with larger molecular weights exhibit greater mobility than those with smaller molecular weights?

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kindly reply me. Thanking you in advance.

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