Annealing temperature in RT PCR - Positive control differes from target gene?

The primer annealing temperature , after the gradient PCR analysis, for the target gene differs from the optimal annealing temperature in housekeeping gene (positive control). What to do ? The...

06 July 2019 7,073 4 View

Fish DNA Isolation , problem with DNA degradation ?

After several protocols for gDNA isolation i have noticed that in all samples teh DNA is totally degraded. I have used several papers, some of them...

02 March 2017 9,220 7 View

RP-resolving power of primer?

08 September 2016 3,351 0 View

Why do I get UREA PAGE GEL smeared bands?

11 December 2015 8,898 14 View

Why does my diagnosis restriction digest not work when PCR verification of my insert works?

08 September 2014 2,133 5 View

Any suggestions for Brucella isolation?

I am in the middle of my project- expression of U-OMP16. I am very pressured for time, and cannot wait for a fresh sample of infected animal with brucellosis in order to isolate the Brucella...

11 December 2013 7,323 0 View

Can someone suggest where to find anti-Omp16 mAbs ?

Monoclonal antibody

08 September 2013 9,265 0 View

Darwin Software - pylogenetic tree construction based on allel number ?

Respected, Does anyone know how to construct a phylogenetic tree base on (PIC) and heterozygosity (H) values calculated for each SSR primer using Darwin software ? The PIC and heterozygosity...

01 January 1970 4,481 13 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