Kunal Chopra

13 Questions 11 Answers 0 Followers

Questions related from Kunal Chopra

What is the best buffer recipe for genome extraction from tissue?

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 544 1 View

Why do high fidelity polymerases remove/not leave any 3' overhangs on PCR products?

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 9,508 5 View

Can the same ELISA assay kit for a hormone work across all species?

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 3,607 2 View

Can we do total RNA extraction from tissues preserved in 4% PFA?

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 8,113 4 View

Is the first ATG in exon 1 always the start codon?

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 3,454 9 View

Has anyone used Levothyroxine for treatment of hypothyroidism in an animal model?

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 6,332 2 View

What is a good strategy to design CRISPR gRNAs?

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 1,054 13 View

How does one interpret bands in negative controls for PCR?

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 8,326 10 View

Can choice of gene fragment influence the success of PCR?

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 769 2 View

Can primers degrade?

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 1,886 3 View

How can I increase cDNA yield from reverse transcriptase PCR?

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 7,908 8 View

Are you familiar with restriction digest with a single enzyme?

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 3,152 12 View

What is the ideal ratio of insert to vector when doing cloning?

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 4,975 6 View