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...

28 September 2016 3,530 3 View

Does anyone have an experience with sucrose cushion centrifugation for virus?

I try to purify virus-like particle that express in recombinant yeast. By sucrose cushion, then subject to equlibirum density CsCl centrifugation. After I culture yeast for 100mL, harvest, break...

20 April 2016 1,139 3 View

Does anyone use re-boiled Salmon Sperm DNA for Yeast LiAc transformation and get a low efficiency?

I use Lithium acetate transformation method for transforming intact circular plasmid into yeast. In past, I always aliquot a new salmon sperm DNA from commercial tube, then boil in water for 10...

25 August 2015 1,693 8 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...

24 June 2015 3,557 3 View

Where can I get L1 HPV52 and L1 HPV58 antibody?

17 November 2014 6,897 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...

26 October 2014 6,610 7 View

Should I clone these genes or synthesize them because I have less time?

I do a M.Sc. thesis about expression of recombinant protein in Hansenula polymorpha (syn. Pichia angusta). Since there is no commercial vector available, we have to construct it ourselves. I have...

03 September 2014 7,131 8 View

Could you help me to find the codon usage table of Hansenula polymorpha?

Now, I'm doing my thesis about expression of recombinant protein in Hansenula polymorpha.Then, I have to do codon optimization of a gene in H. polymorpha, but I cannot find codon usage table of H....

08 July 2014 156 6 View

Looking for Materials with high thermal expansion coefficient and high temperature resistance?

03 March 2021 8,272 1 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

How to grow E.coli on the low salt LB agar plate with 50 or 100ug/ml of Blasticidin?

Hello, I would like to ask how to grow E.coli which resists 50 or 100ug/ml of Blasticidin. I have tried many methods by using low salt Luria-Bertain(LB) agar plate. However, no colonies grow. Let...

01 March 2021 2,027 2 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

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