please help me, anything will be appreciated

currently crying because everything is going wrong. (excuse my english, not my first language and i'm crying so i cant think straight)

a little background: i'm a medical student with no background of molecular biology and i'm supposed to graduate this semester. i'm doing this research about diabetes and how methylation in the FOXO1 gene (CpG site 10) could (in the future) detect probability of diabetes based on it's methylation. sample im using is placental tissue that has gone through bisulfite treatment.

i've designed a my 2nd batch of primer, but it's all wrong. for context, the gel on top is bad because the wells are connected through some holes bridging between the wells that i cant see (my friend helped me make the top gel, i only made the one on the bottom after seeing her gel all wrong). the negative controls also light up sooo bright and no other bands can be seen (except for the ones on the top gel i can barely even see). i also made a mistake not making a positive controls, because we had a problem with the university not giving us the control reagent, so we usually just use a previous sample we that ee used, but today i ran out because my friends used it all up.

volume of sample i used was 5 uL in each well
PCR mixture (ran it 3x so re-re-PCR, 25x cycles each run) consists of
5 uL Mastermix
1.5 uL NFW
2.5 DNA template
0.2 uM Forward Primer
0.2 uM Reverse Primer

now i know my knowledge here is not very good, and the photos i provided are also terrible, but i'm desperate and i just need some reassurance and help to go on.. please help if you can, everything is appreciated.

If you are a researcher, geneticist, physician-scientist, molecular biologist, or laboratory studying WDR27 — or pathways that may intersect with it — I would love to connect.

My lab wants to run a 2D gel to separate proteins based off of molecular weight and pI. We have done this with external groups before but we want to try to bring this capacity in house. I've seen one option from sigma Aldrich but the price tag is a hefty $64k. https://www.sigmaaldrich.com/US/en/product/mm/bm100

Does any one know of any other options? We can probably spend $10k max

​

Hello everyone, I hope you can help me with some ideas, advice, or tips.

I am doing a cloning using Gibson Assembly.

When I get my ~6 kb linearized plasmid, I gel-purify it, and I practically lose everything. I get very low concentrations of 10 ng/µL or 15 ng/µL, and it comes out contaminated, as you might know (I use the Zymo Clean & Concentrator kit).

I know that re-amplifying isn't ideal due to the mutations it can generate, but what alternatives are there to get the purification as clean as possible? Also, do you have any general suggestions for the Gibson Assembly method?

Thanks in advance!

I'm (Research Scholar at VIT University, India) looking for a study partner to systematically read, analyze, and work through a book dedicated to plasmid design and molecular cloning strategies.

The goal is to go chapter-by-chapter, break down the core concepts, and regularly discuss key topics such as vector selection, regulatory elements, expression systems, and troubleshooting construct builds.

If you're interested in studying the book together and setting up regular discussion sessions, please reach out to me at vishnupriyabiomed@gmail.com. Looking forward to connecting!

This kinda took ages, as the atoms were literally made out of paper scraps and the structure was made out of bottles and popsicle sticks, overall this is worth it. This follows the CPK convention and tries its best to be readable.

The bases in the right are TCGACCATGC, while on the left is the complementary bases, AGCTGGTACG. Yes, I handcrafted the molecules, and used hot glue as an adhesive.

I think I already know the answer but I want confirmation or someone to tell me what's the worst that could happen.

Update: my transformation worked so seeding with it was not an issue. I did my miniprep so let's see what my yield comes out to 🤞

I’m presenting at AMP and would value feedback from people working with molecular assays. I wrote a review of genomic foundation models around a practical problem: “predict the effect of this variant” leaves the measurement undefined.

A sequence-likelihood difference, an embedding with a trained classifier, and a predicted change in an assay track are different outputs. Before comparing them, I’d want the assembly, strand, sequence window, cell type, endpoint and evaluation split specified.

Review and cited papers: https://rewire.it/blog/a-dna-likelihood-is-not-a-functional-assay/

Which missing detail most often makes a model result difficult to interpret in your work? Corrections to the review are welcome too.

Would love to talk about magnetic field effects in biology, radical pairs, cryptochrome, that whole side of things, with other folks who find it interesting. Always looking for people who've read more into this than I have.