Is Qiagen's Rotor Gene Q machine any good?

04 May 2015 2,658 6 View

Can you import TIFFs into BIORAD ImageLab software?

We recently got a new BioRad digital imaging western blot detection system. All is fine except that the software (ImageLab) can export images to TIFF but then refuses to import them again for...

06 July 2014 965 9 View

QPCR data - Positive/negative fold change or relative expression ratio?

I would be interested in peoples opinion on the best way or presenting qPCR data. I often present data as relative expression, thats is relative to 1. Here a ten fold increase is plotted as a...

04 May 2014 3,043 24 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

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

Best plasmid could be used as a standard curve for AAV titration?

I have been trying to have a good AAV titration for my production and I have tried different plasmids to set up as a standard curve but each one behaves differently. i am trying to use ITR for...

23 February 2021 5,167 3 View