Is it possible to replace yeast nitrogen base with other materials?

I am going to do induction in Pichia pastoris. But my lab does not have yeast nitrogen base. Just wondering if I can substitute with some other things.

09 October 2014 9,345 4 View

Would I be able to see a difference between a induced and uninduced empty pET vector?

Hi people, I am having a lot of difficulties in my protein expression. I am wondering if my induction has failed to work? Too bad, i dont have any positive control. So i am using the empty...

09 October 2014 8,610 9 View

Gene with high GC content is difficult to be expressed in E. coli. How do you define high here?

People I need some help here.

09 October 2014 8,764 0 View

Can anyone recommend a website that can predict RNase E cutting site?

Do you know of any website which would predict RNAse E cutting site? or how to predict it? We are having problems expressing a foreign mRNA in E.coli and one possibility was that the mRNA was...

09 October 2014 3,617 3 View

D-lactose monohydrate versus alpha lactose monohydrate for use in autoinduction media

I need lactose for my autoinduction media for E.coli. Just wondering if I could use the D-lactose monohydrate because I can't find any alpha-lactose monohydrate in my lab. Are they the same?

08 September 2014 5,368 8 View

Anyone have experience in autoinduction?

I have cloned lipase gene into pET28+ and express it in Rosetta(DE3)pLyss. I have tried to induce with IPTG but I can't spot any difference between the clone and the empty pET. I varied the...

08 September 2014 5,478 4 View

How does one extract inclusion bodies from E. coli?

what is the most effective method here

07 August 2014 8,345 2 View

Can anyone help with induction in E. coli?

Dramatic induction was observed, but no desired protein band was observed. I run an empty pET as a control. There doesnt seem to be difference with the empty pET and pET with insert. I have no...

07 August 2014 3,715 13 View

How do you choose a good antibody for western blot?

I have cloned my lipase gene from rice in pET-28(+) His-tag vector. Was wondering how do I choose/design the primary and secondary antibodies for western blot. I am totally new to this. Was hoping...

06 July 2014 1,768 4 View

Could anyone help me with my protein expression protocol?

Protein Expression in Prokayotic (Rosetta (DE3) E. coli) (1) Transformant Rosetta (DE3) cells with recombinant pET28(+) were grown on LB/Kanamycin/Chloramphenicol agar plates overnight. (2)...

06 July 2014 1,501 6 View

Can linear DNA be used as template for site-directed mutagenesis?

Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...

03 March 2021 401 4 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

Which is a lucid standard comprehensive book to guide on psychological scale construction and its standardization?

02 March 2021 7,082 3 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

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 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

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View