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

How bad is this overlap primer with 2 sets of sextet adenine?

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...

31 December 2019 3,921 3 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

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

Buffer to disrupt platelet alpha granules for PF4 quantification?

Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...

03 March 2021 1,499 2 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

How to increase a model accuracy ?

dear community, my model is based feature extraction from non stationary signals using discrete Wavelet Transform and then using statistical features then machine learning classifiers in order to...

03 March 2021 6,994 5 View

How optimize the Size Exclusion Chromatography?

Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...

01 March 2021 2,215 2 View

Why are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 View

What do I do with DLS peaks that are from the buffer?

I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak...

01 March 2021 9,015 2 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable resuspension buffer for TCA/acetone precipitated protein?

I need to extract protein from fermented carbon sources for Bradford assay. Most researchers experiencing insolubility of pellet in resuspension buffer. Please assist me to select most suitable...

26 February 2021 7,129 3 View

Is there any one who can provide a donation of HPV detection and genotyping kits in exchange collaboration fors?

Warm greetings My name is Ayichew S. and I am doing my Ph.D. in Medical Microbiology (on Human papillomavirus). At this stage, I have almost completed a clinical sample Cervical swabs)...

25 February 2021 1,660 3 View

Is there any one who can provide a donation of HPV detection and genotyping kits in exchange collaboration forms?

Warm greetings! My name is Ayichew S. and I am doing my Ph.D. in Medical Microbiology focusing on Human papillomavirus). At this stage, I have almost completed a clinical sample (Cervical swabs)...

25 February 2021 3,612 3 View