Pramod Bhatt

5 Questions 6 Answers 0 Followers

Questions related from Pramod Bhatt

Adding more media to bring down O.D before protein induction?

Hello folks, I found that my E.coli BL21 DE3 star cultures grew to O.D. 1.2 before I could induce with 0.5 mM IPTG. I normally induce at O.D. between 0.4 to 0.8 when they are in exponential...

08 August 2018 5,782 0 View

White cloudy precipitate during Ni-NTA purification?

Hello folks, I had a N terminal His-GST-TEV cleavage site tagged protein (15mg) on dialysis over ~40 hours in the presence of TEV protease (1mg) with a buffer change in between. Buffer is 50 mM...

07 July 2018 1,311 5 View

Leaving His-tagged protein bound to N-NTA beads in wash buffer overnight?

Hello folks, I am performing Ni-NTA purification of N-term His-GST-TEV cleavage site -tagged protein. Due to a time limitation I need to leave it overnight in wash buffer (NaH2PO4 pH 8.0, 300mM...

07 July 2018 4,868 4 View

How to identify degraded RNA and non-denatured RNA products on a PAGE-UREA gel?

When running RNA on a Urea-PAGE gel, how would you differentiate between degraded RNA from non-denatured RNA? i.e., does a smear imply degradation/overloading and sharp non-specific bands imply...

09 September 2016 1,674 0 View

Unusual Chloramphenicol absorption in sucrose gradient on Isco fractionator?

Hello there, I have prepared 10-55% sucrose gradients containing 1mM Chloramphenicol and run them on an Isco sucrose gradient fractionation system, but the absorption is too high to record on the...

04 April 2016 9,895 3 View