14 Questions 154 Answers 0 Followers
Questions related from Amy Johnson
Hello - I am studying this protocol which claims to use pcr to clone in/replace a small piece of an sgrna plasmid with a new guide sequence provided by an oligo with homology to the sgrna...
03 March 2020 6,453 2 View
I am using the following protocol from a CSH lab manual edited by Doudna on CRISPR, to attempt to retarget sgrna scaffold in a plasmid. but....I get DNA stuck in the well, not migrating....
02 February 2020 6,424 3 View
I have been beyond meticulous with NEB Builder, and it has never once worked. I have had success with OE PCR, but NEB Builder fails totally, every time. I get nothing but smear and concatenation...
02 February 2020 9,100 9 View
I ran a gel where what is almost certainly my 8,700 bp plasmid appears to be running slightly heavy versus the 1kb DNA ladder bands. I have been advised at these extremes, the ladder isn't highly...
02 February 2020 9,326 7 View
When I use https://tmcalculator.neb.com/#!/main I often get this message with a recommended annealing temperature of as high as 64 degrees "Why is this annealing temperature so high?...
01 January 2020 337 4 View
Neb Builder suggested this primer to me for PCR overlap ligation, and I appear to be stuck with it. At least it isn't 6 guanines! How much trouble do you think this bad forward primer could...
01 January 2020 4,015 3 View
This paper Danquah, M. K. & Forde, G. M. Growth Medium Selection and Its Economic Impact on Plasmid DNA Production. Journal of Bioscience and Bioengineering 104, 490–497 (2007). Makes the...
12 December 2019 6,224 6 View
I am following alkaline lysis protocol very carefully, with special attention to the correct pH of all the three solutions - TE with glucose, NaOH with sds and K-acetate with glacial acetic acid...
12 December 2019 4,855 7 View
I subcloned the first two gibson fragments after a fresh gibson ligation, the products are joined from an 800 bp promoter and a 1,500 bp gene. I see that band. That band appears in the right...
12 December 2019 1,396 9 View
is this Gibson assembly primer set generated by NEB builder sane? Hi everyone, I am trying to assemble a gene into a cassette with a left and right homology arm (added those to the gene...
11 November 2019 9,345 3 View
Hello: I was wondering if I would encounter problems with simply using PCR to amplify a fragment of a plasmid that is mixed with the raw genomic DNA from a bulk phenol:chloroform extraction of a...
11 November 2019 1,964 14 View
Hi: I was wondering if running a phenol:chloroform extraction before an alkaline lysis separation of plasmid dna from genomic would be of benefit (ie cleaner sample free of protein etc) or would...
11 November 2019 4,903 5 View
Hey: I am thinking that sodium acetate would be equivalent to potassium acetate in a neutralization buffer to be used after alkaline lysis of bacterial cells for plasmid extraction. The recipe...
11 November 2019 2,420 4 View
I have qubit confirmation that my pcr produced 21 ng/ul of dsDNa in a 50ul reaction volume. I chose primers for a specific gene/sequence of 1.4kb . But my gel electrophoriesis shows a nice...
10 October 2019 686 14 View
