Why do I receive completely different sequencing result?

Dear all, I cloned 25bp fragment into a vector. My no-insert ligation has no colonies and my insert+vector ligation has many. Which is the first clue that I have cloned the fragment. Than I...

11 December 2019 7,539 8 View

How can I eliminate yeast contamination?

Dear friends, We kindly get U-251 MG cell line as a gift from an other lab. after 3 passage we have determined that cell line has yeast contamination. Each stock is contaminated while other...

11 December 2018 5,313 8 View

How Can I detect artificial miRNA in stable cell lines?

Dear all, I have generated Stable Cell lines via G418 selection. I have used pCMV-miR vector.Except Neo-GFP region, my chance is 2/5 to integrate genome without damaging CMVpromotor-insert-PolyA...

07 August 2018 8,094 2 View

Is it possible to clone without PolyA signal?

hey, I have generated stable cell lines with random entegration of pCMV-miR vector. Vector contains a neoR gene fused with GFP with IRES. I got some GFP+ and GFP- colonies. and GFP- colonies...

07 August 2018 612 3 View

G418 selection cell death problem?

Dear Friends, I performed a kill-curve experiment and determinated that ; at 400mg/ml g418 consantration only 5-10 non-profilable cell survived at 7 day in 48 well-plate, plated with 50.000 U87...

05 June 2018 7,987 3 View

During luciferase assay my miRNA target luciferase gene's coding sequence?

Hey, I have a small problem. I have cloned my target gene's UTR into pmir-Glo vector. But the problem is that frefly luciferase gene also contains miRNA target sequence. Thus when I clone my UTR...

04 May 2018 9,073 0 View

Transfection efficiency as well as transgene expression in high passaged GBM cell lines?

Dear all, I am working with U-87 MG (a glioblastoma cell line whose origine is unknown but male) and T-98G. I have clonned my miRNA genes into pCMV-miR vector that express transgene via CMV...

03 April 2018 6,728 3 View

How to eliminate low transfection efficiency?

I am working with glioblastoma U-87 cell lines. I have pCMV-miR expression vector ~8kb. I tired both Transfast, Fugene HD, PEI, Jetprime. They had max %30 efficiency. Transfast best with 1:1...

03 April 2018 9,602 8 View

Can 1 kb genomic fragment be used as pri-miRNA?

Hey all, To overexpress a miRNA; instead of pre-miRNA sequences, pri-miRNA sequences are also being used driven by RNAP II promoters. in articles these fragments nearly miRNA+100-300 bp flanking...

03 April 2018 3,764 5 View

Why do I have colonies after 24h on kanamycin plate with no insertion?

Hey, I am trying to clone a 600 bp fragment into pcmv-mir vector. I use 30mg/L kanamycin in my agar plates. I digest 1 uq plasmid with 10 unit of enzyme (Sgf I and MluI) over night. I run a...

02 March 2018 4,734 7 View

Can we classify HV and LV lines based on line length?

I have dataset which shows the length of power lines. I need to classify the lines based on the line length. Is there a rule to classify the High voltage (HV) and low voltage (LV) lines based on...

03 March 2021 4,116 4 View

Buffer to disrupt platelet alpha granules for PF4 quantification?

Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...

03 March 2021 1,499 2 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

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02 March 2021 3,060 3 View

How optimize the Size Exclusion Chromatography?

Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...

01 March 2021 2,215 2 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Why are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View