My DNA bands have donut shape on Agarose gel electrophoresis?

16 December 2024 0 1K Report

Hi, I move to a new lab. And I found that my DNA bands show donut shape on Agarose gel electrophoresis. I can't slove this problem. I use 1X TAE buffer. Run 100 V for 30 min. Post-staining gel with Fluorescent dye. 1X commercial loading dye. All with type 1 water. I change many brands of commercial DNA ladder. I try to optimize many factors, but it not succes. Can you please suggest me?

Badges
Science method

More Tatpong Boontawon's questions See All

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 3,566 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 1,219 3 View

Does ethanol denaturing enzyme, and causes non-function of protein?

I use Pichia pastoris for recombinant Lipase enzyme from Candida sp. production (secreted into medium). I try to follow the graduated members that do this project, unfortunately the protocol not...

04 March 2016 8,715 4 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 1,838 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 3,731 3 View

Can S. cerevisiae URA3 gene can function in H. polymorpha NCYC495?

I constuct plasmid that contains FMD promoter, MOX terminator, HARS1, and URA3 gene from S. cerevisiae, then transform by LiAc method to H. polymorpha NCYC495 for three times. But every time don't...

25 May 2015 8,029 3 View

Can intact circular plasmid integrates into Genome of Yeast S. cereivisiae?

I want to design an experiment to confirm that my S. cerevisiae URA3 gene can function in S. cerevisiae. My plasmid that is a BluescriptiiSK+ harboring S. cerevisiae URA3 gene (and don't has any...

22 May 2015 3,729 6 View

Does anyone suggest pBluscript II SK(+) or related with Kanamycin resistance marker

Does anyone suggest a plasmid that be a "pBluescript II SK(+) with Kanamycin resistance marker (instead of AmpR)" or relates. Best regards, Tatpong

02 December 2014 8,769 3 View

Where can I get L1 HPV52 and L1 HPV58 antibody?

I'm doing my M.Sc. project in the title "Expression of L1 HPV52 and L1 HPV58 protein by yeast" but I can't find any antibody use for detection these proteins. Can anyone tell me. How can I find...

17 November 2014 6,936 3 View

Has anyone used H. polymorpha NCYC495 for recombinant protein expression?

I try to find yeast H. polymorpha that have auxotrophic markers in many collections for use with my M.Sc. project, but I found only H. polymorpha NCYC495 ura3- or leu2- available. I read many...

10 November 2014 5,028 3 View

Does anyone know what might be causing the smeared bands on my western blot?

I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...

10 August 2024 7,480 3 View

Why is my ASO degraded quickly on agarose gel?

I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...

06 August 2024 3,130 2 View

Low-yield gel extraction problem?

I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...

05 August 2024 9,798 3 View

I'm optimizing a tetra-ARMS PCR protocol with amplicon sizes: 165bp, 123bp and 93bp. Why, in the gel, only 165bp is visible while I'm expecting all 3?

It's an end-point PCR protocol. I'm using 1.5% agarose gel with SyBR Safe dye and TBE as a running buffer, visualization on BioRad XR+ system. I was primarily thinking of primer efficiency,...

01 August 2024 4,673 4 View

Transfection in HEK293T cells?

Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...

31 July 2024 9,892 4 View

PCR showing no bands - Master mix and primers didn't mix?

Hello everyone, I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for...

31 July 2024 2,406 6 View

How to check pH of a high strength hydrogel?

We are working on biopolymeric hydrogels. Our system is highly viscous and sticky, and the gel formed are high in strength. We are unable to use pH electrode and pH strip. Please suggest an easy...

30 July 2024 942 2 View

Following click reaction in cell lysates, protein is immobile and remains at the top of the gel in SDS-PAGE?

I am using CuBr/THPTA for a click reaction in total cell lysates. I am facing issues with my protein sample in non-reducing SDS-PAGE where it's not migrating properly and most of it remains at the...

29 July 2024 950 4 View

How to retain the a GFP tagged gene expression in stable cell line?

Hello , I established a stable cell line expressing GFP tagged to a centrosomal gene having G418 drug selection marker. I validated the stable line by IFA and Western blotting, results are fine....

29 July 2024 5,007 0 View

Is it necessary to attach fluorophore at 3' end in MIC RT PCR machine for Dual hybridization Probe designed especially for genotyping assay?

I designed 2 dual hybridization probes for my single SNP. where I attach my fluorophore 5'-FAM-nnnn-BHQ1-3', and 5'-HEX-nnnnnn-BHQ2-3'. I need to run it on the MIC RT PCR machine by Biomolecular...

28 July 2024 5,658 0 View