Shilpi Kumari

14 Questions 4 Answers 0 Followers

Questions related from Shilpi Kumari

Why does my time constant show 2.2ms after electroporation of E. coli cells?

I am trying to transform my plasmid into electrocompetent E. coli cells. They were prepared by washing the cells four times with sterile ice-cold water. After the pulsing, the time constant was...

15 May 2023 8,659 6 View

How do I induce protein expression at 42 degree celsius without a shaking water bath?

I have a temperature-sensitive plasmid that gets cured at 42 degrees and simultaneously expresses a protein under the regulation of a temperature-sensitive lambda repressor. For its curing and...

09 May 2023 6,863 1 View

Do chemically competent E. coli cells show the same efficiency as the electrocompetent E. coli cells?

I have to perform the genomic integration of a gene, would it be better to use E.coli cells made competent by chemical method or by electroporation? Thank you

24 April 2023 6,755 4 View

Is there a problem if there are 2 additional bases between the promoter and the gene?

I have to clone a gene below its native promoter but it is part of an operon, so I need to amplify the two genes separately and clone it in two steps. I found a plasmid in my lab which already has...

24 April 2023 6,491 5 View

Has anyone overexpressed a membrane protein after addition of 6X His tag?

Can the addition of 6X his tag lead to a significant decrease in the specific enzyme activity?

24 April 2023 8,627 3 View

Is it possible to clone a fragment as short as 31 bp from genomic DNA?

I need to clone the lac promoter from the E. coli K-12 strain, which is 31 bp long.

07 December 2022 9,373 3 View

How can I look for a strain on CGSC which is lacZ-?

I have to make lacZ- revertants of E. coli MG1655. I first need a strain which is lacZ-. How can I find a strain with this genotype on the Coli Genetic Stock Center (CGSC)?

04 August 2022 4,198 0 View

Which Polymerase should I use for obtaining a gene through PCR which is 3Kb long?

The gene of interest is 3Kb long and I wonder if Taq Pol would be suitable for it as it can incorporate the wrong bases. Should I rather go with Pfu Polymerase?

05 July 2022 8,856 3 View

Why am I getting different sizes of colony on my LB-Agar plates?

I am trying to streak the K-12 MG1655 strain on LB-Agar plates and there is a lot of heterogeneity in the size of the colonies. When I pick up 1 discrete colony and transform it, even after...

04 May 2022 341 2 View

Why is GST tag used?

We know that GST has a large protein size, 225.5 KDa. When other tags are available, say 6X His tag, then why is a tag with such a huge molecular weight used which might interfere with downstream...

24 May 2021 8,903 7 View

What is the concentration of PCR buffer in the GBiosciences Taq Polymerase 2X mastermix?

I am working on PCR and I am unable to understand the buffer concentration in the premixed 2X mastermix of GBiosciences. Is it 10X or 2X?

18 March 2021 1,890 2 View

How can I screen for false positives in PCR?

I am getting a band in negative control as well and so I want to check whether the other bands I am getting are positives or false positives.

18 March 2021 8,009 3 View

Why am I getting a band in my negative control in colony PCR?

There is a band of same size as that of my insert in the negative control PCR reaction,though it is much less intense than the other colonies. What could be the explanation?

11 March 2021 8,904 6 View

What is the working concentration of EtBr?

I found different stock concentrations, e.g., 10mg/ml and 5mg/ml but unable to find the working concentration.

18 February 2021 6,199 3 View