27 Questions 17 Answers 0 Followers
Questions related from Kunal Chopra
I want to target a gene in the zebrafish via CRISPR. This gene has 3 protein coding splice variants. There are some shared regions across all variants in the the first intron. Does anyone have...
21 October 2019 2,969 2 View
I want to count pigments on AB strain zebrafish and I've been looking at literature for the same. Epinephrine appears to be the chemical of choice but Sigma Aldrich labels it as highly toxic....
03 August 2017 3,908 0 View
I have dissolved L thyroxine (Sigma) into a solution of NH3 in methanol (sigma). I was wondering though if there is a desirable temperature at which a solution containing the dissolved hormone can...
12 July 2017 8,996 2 View
I need to dissolve T4 and the accompanying notes advise to dissolve it in 4M NH4OH in methanol. Perhaps NH4OH is a common reagent or maybe not but either way, I don't have any in the lab. Can NaOH...
20 June 2017 1,908 4 View
I work with zebrafish adults and larvae and am trying to characterise mutations from CRISPR and ENU mutants. I either extract the genome from entire larvae between 3 and 5dpf or adult fin clips....
02 April 2017 636 1 View
I have been working with a high-fidelity polymerase (ExTaq's TaKaRa) and I had challenges ligating these PCR products into the pcr 2.1 vector. I then found out that TaKaRa does not leave any 3'...
09 March 2017 9,564 5 View
I am testing zebrafish and Xenopus laevis for reduced hormone levels. It is the same hormone in both species. My method of choice is ELISA. However, when I researched for the available kits, they...
30 August 2016 3,715 2 View
Speaking of model organisms including mice, Xenopus and zebrafish, would a total T4/T3 test be a better indicator for hypothyroidism over free T4/T3? Also, out of T4 and T3, which would be a...
07 August 2016 5,754 5 View
I have been doing a lot of total RNA extraction from wild type zebrafish. However, with other experiments going on simultaneously, I often find very little time left for dissecting the fish and...
06 July 2016 8,208 4 View
I am using the TriZol method for extracting total RNA from adult zebrafish tissue, whole Xenopus larvae and whole zebrafish larvae. Considering that my 260/280 and 260/230 values are near about...
28 June 2016 4,416 8 View
I am interested in extracting RNA from eyes, liver, intestine and gills of adult zebrafish. Following TriZol extraction, I run 500ng on a gel and sometimes I get nice strong bands whereas at other...
21 June 2016 1,349 3 View
I am designing CRISPR gRNAs and wanted to check if it is usually safe to assume that the first ATG in exon 1 is always the start codon. The model organisms I am working with are zebrafish and...
06 June 2016 3,505 9 View
I have a mutant zebrafish strain that likely has hypothryoidism as a result of the mutation. I am basing this on the fact that the adults are dwarfs compared to wild type fish and also do not...
31 May 2016 6,421 2 View
I just did a total RNA extraction using TRIZOL and the 260/230 values of all samples is between 0.50 and 0.90! I also noticed that for some of them the pellet did not dissolve completely. Also, I...
27 April 2016 5,520 7 View
I am doing so tail regeneration experiments on Xenopus laevis and Xenopus tropicalis. I have noticed that their tails always regrow at an angle instead of straight. Is this the norm or could I do...
09 April 2016 753 4 View
I am trying to design CRISPRs for a couple of selected genes in zebrafish and Xenopus tropicalis. So far I have used mit.edu, crisprscan.org and chop chop. Zebrafish gRNAs have been especially...
04 April 2016 1,121 13 View
Hi! I am interested in doing cell movement and tracking experiments in wild type and transgenic zebrafish larvae. Essentially, I subject the larvae to stimuli and study the movement of cells that...
08 February 2016 1,338 8 View
I did a PCR for cDNA from 3 different tissues, 2 of which do not express my gene of interest. While only the predicted tissue showed the band for my gene of interest, the negative controls for...
25 January 2016 8,468 10 View
Lately, I am being plagued by a hit and miss situation concerning the reproducibility of my PCR. I am trying to check expression of a gene reported to be expressed in the zebrafish intestine. I...
05 January 2016 830 2 View
I have been struggling for over 2 months with a PCR. I am trying to check if a gene is expressed in the intestinal tissue of zebrafish. Following total RNA extraction with Trizol, I do a DNAse...
04 January 2016 1,953 3 View
I am trying to obtain cDNA from RNA samples from 5 different tissues. Currently, I am doing the following: 1ug RNA + random oligomer (50uM) + water followed by 10mins at 70 degrees C. Then I add...
07 December 2015 7,958 8 View
I believe this may be directly related to concentration of primers used, but like dNTPs, can primers also get used up fully in the course of a PCR? I am using primers for a housekeeping gene at a...
29 November 2015 7,315 3 View
I would like to undertake a thyroid treatment and monitoring assay in Xenopus tropicalis. I am new to using this is a s a model organism and would like to know if there are any classical landmark...
09 October 2015 8,931 2 View
I am looking to do a T4 assay in some mutant zebrafish who appear to show stunted growth. I am interested in observing whether treatment with T4 is going to have a positive effect and if these...
06 October 2015 7,371 0 View
I am cloning out a fragment, using SacII, containing the ORF of my interest and cloning it into a SacII site in the multiple cloning site of my vector. The insert can obviously orientate itself in...
09 April 2015 3,206 12 View
I am working with several large Gateway clones (upto 25kb), many of which are proving a challenge to be amplified via midi prep but work fine for mini preps. I need to inject these into zebrafish...
05 February 2014 5,137 3 View
I have been trying to clone a ~2.5kb insert into an ~8kb vector. I have been given varying advice and to make things worse, the cloning hasn't been working. I want to start all over and identify...
01 August 2013 5,042 6 View
