If I coat protein for indirect ELISA overnight , Should I add PMSF into coating buffer or not?

I express recombinant protein in yeast. After that harvest cell and resuspend cell in Phosphate Buffer Saline (PBS) buffer with 1 mM PMSF (as protease inhibitor), then break it by glass bead and...

08 September 2016 6,964 3 View

Why In Vitro (Cell-free) recombinant protein synthesisis not used instead of microorganisms in the industrial scale?

11 December 2015 7,865 9 View

Could anyone suggest the Deletion of which gene can reduce hyperglycosylation in Pichia pastoris?

In yeast, we face to the problem of recombinant protein expression because of its ability in hyperglycosylation. If I aim to reduce the hyperglycosylation without affect to the growth of the Yeast...

05 June 2015 7,421 4 View

Can S. cerevisiae URA3 gene can function in H. polymorpha NCYC495?

I constuct plasmid that contains FMD promoter, MOX terminator, HARS1, and URA3 gene from S. cerevisiae, then transform by LiAc method to H. polymorpha NCYC495 for three times. But every time don't...

04 May 2015 166 3 View

Could anyone suggest why I get very little amount of plasmid DNA after extraction from E. coli.?

I already obtained two plasmid that harboring gene synthesis A or B. First is pUC57 harboring gene A (size of insert 1.5 kb). Second is pUC57 harboring gene B (size of insert 1.6 kb). Then, I...

03 April 2015 7,851 14 View

Does anyone ever use PDM (Plasmid DNA Medium) for E.coli plasmid preparation?

I read from Bitesize Bio website that Michael K. Danquaha, and Gareth M. Fordea (2007) published a paper for plasmid DNA production. This PDM can give a higher yield than the other E. coli medium,...

02 March 2015 4,849 3 View

What is the chemical for washing and removing an undesired or contaminated Plasmid DNA?

Now, I'm in the step of plasmid construction in E. coli. But I found a big problem because of an undesired or contaminated plasmid DNA (some one called Ghost plasmid) in my transformants (more...

01 February 2015 9,007 6 View

Does anyone suggest pBluscript II SK(+) or related with Kanamycin resistance marker

Does anyone suggest a plasmid that be a "pBluescript II SK(+) with Kanamycin resistance marker (instead of AmpR)" or relates. Best regards, Tatpong

11 December 2014 1,574 3 View

Where can I get L1 HPV52 and L1 HPV58 antibody?

10 November 2014 3,820 3 View

Could you suggest high fidelty DNA polymerase for cloning about 300-600 bp DNA fragments?

I want to clone about 300-600 bp DNA fragment from yeast genome. Since there have many high-fidelity DNA polymerase i.e. Phusion, KOD HiFi, Q5 etc. Colud you suggest me which is the most proper...

09 October 2014 3,546 8 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

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

I want to performe MALDI-TOF MS for bacterial identification, I would appreciate it if anyone can tell me the matrix solution preparation protocol ?

based on what I've studied on multiple papers, for peptide and protein identification Alpha-cyano-4-hydroxycinnamic acid (CHCA) is an appropriate matrix to use, I also know that the CHCA powder...

26 February 2021 6,799 3 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