Does antibiotic affect the growth rate of resistant bacteria too?

If a bacteria bearing the resistance gene for an antibiotic is grown in presence of the antibiotic, how slow will it be compared to its native form (where it does not have the resistance gene...

06 July 2017 683 8 View

Why is RPMI media's colour is changing so frequently?

I'm using RPMI 1640 media from Himedia. Within 24 hours the colour of the media is changing to yellow even with very low density of cells. I have checked for any kind of contamination, but that is...

04 May 2017 1,335 14 View

What should be the ideal time for batch binding of protein to affinity tagged resin?

I have to purify a bacterial protein from E. coli. My protein's yield is quite low. I have read that batch binding gives better yield, if so, for how much time do I have to incubate my protein...

01 February 2017 3,912 6 View

Does restriction digestion also require supercoiled form of plasmid?

Isolated plasmid when mostly in supercoiled form eases most of the downstream processes, but is it essential for restriction digestion also? If there is less supercoiled and more of nicked...

11 December 2016 8,508 6 View

How stable is the antibiotic ampicillin?

I know that beta lactamase produced by the colonies will degrade the ampicillin in vicinity where spurious satellite colonies can grow but how long can unused plates of Luria Bertani Agar media...

10 November 2016 7,828 9 View

Why do genes cloned in same vector, under same promoter have different levels of expression?

I have cloned my gene of interest (WT) in a vector and in the same vector with same restriction sites I have cloned the point mutated gene. I am not getting any expression for the WT whereas the...

09 October 2016 3,977 7 View

To eliminate the nicked circular plasmids from plasmid prep can ligase be used?

In a plasmid maxi prep, many a times, nicked circular species are isolated apart from supercoiled. Can this nick be rejoined by incubating the prep with ligase? I know it won't increase...

08 September 2016 5,444 6 View

Is there any tool available to determine the protein structure?

07 August 2016 4,768 6 View

How stable are N-terminal truncated proteins?

a. As protein folding generally starts from N terminus. If hypothetically I delete some parts of its N-terminus, will it still be stable and able to fold properly? b. If I delete 30 amino acids...

06 July 2016 5,877 8 View

What is the evolutionary basis of DNA Polymerase being able to synthesise only in one direction?

I feel it contradictory to evolutionary advantage that the two strands of DNA as evolved are anti-parallel but the polymerases that synthesize/ replicate them can do so in only one direction....

06 July 2016 3,598 16 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Anyone that can help with details in phosphopeptides enrichment?

Anyone know the amount of protein before enzymatic digestion for phosphopeptides enrichment of serume sample in Ti-IMAC? Has anyone ever use Ti-IMAC(the material is from Chinese company named...

28 February 2021 7,310 3 View

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View