Does this cold spring harbor protocol really edit circular dna with pcr?

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...

02 March 2020 6,358 2 View

Opinions on using PCR to re-target plasmid sgrna, with a "universal" sgrna scaffold?

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....

01 February 2020 6,327 3 View

How accurate are DNA ladders near their 10 kb extreme?

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...

01 February 2020 9,189 7 View

Does NEB Builder EVER work?

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...

01 February 2020 8,962 9 View

Are the very high annealing temperatures from neb Tm software necessary?

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?...

31 December 2019 234 4 View

What are these additional gel bands in my cloning of a Gibson ligation product?

11 December 2019 1,261 9 View

What causes alkaline lysis for plasmid isolation to produce only short dna fragments on gel?

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...

11 December 2019 4,760 7 View

Will adding extra nutrients to LB broth increase plasmid DNA expression?

11 December 2019 6,116 6 View

Sodium acetate versus potassium acetate in a neutralization buffer?

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...

10 November 2019 2,117 4 View

Can I PCR a fragment of plasmid directly from a genomic dna extraction?

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...

10 November 2019 1,873 14 View

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 4 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 View

Research on Supply Chain Management in Indian context?

Hi, I am planning to apply for the PhD degree in the Supply Chain Mgt. with specific area of "Cold Storage warehouses" during Pandemics and wars. Where lock downs and shut downs are frequent....

02 March 2021 285 2 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

How to calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

Anybody with an idea on how do design specific primers for detecting tomato resistance genes tm-1, TM-2 and Tm2^2?

I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There...

28 February 2021 606 3 View

What is the ideal efficiency for a qPCR?

hello everyone, I need to do standard curves for my qPCR, what is the ideal efficiency range? I tried a primer (Mglu2 receptor) that gave an efficiency of 90.2%. Is it accepted?

28 February 2021 1,254 3 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Runs of nucleotides in Sanger sequencing?

I performed site directed mutagenesis, transformation, and then I sent out plasmids for Sanger sequencing and found out that there is extension of DNA just before the stop codon. I am not sure...

27 February 2021 547 3 View