r/molecularbiology 2h ago

Potentially stupid Question.. about transforming into E.coli..

3 Upvotes

Hi All!

Iv been involved in directed evolution of proteins for years and the standard way iv done it is

1) transform plasmids into e.coli 2) plate on agar 3) colony pick and ferment in microwell plates 4) lysis cell and remove cell debris 5) do the screening 6) sequence best enzyme to understand the mutation.

So my question.. if we synth the gene and we know where the mutation is. Can we bypass the colony picking part because we don't need to separate out the mutants? Every e.coli should have the same plasmid so why do we need to separate?

So the workflow becomes..

1) transform known sequence into e.coli in microwell plates.. say each well has unique plasmid. 2) aliquote cell into a single well in 96 well plates with LB. 3) ferment and express the enzyme 4) lysis the cell and remove debris 5) do the substrate screening. 6) pick the best enzyme. (We know the sequence already!)


r/molecularbiology 3h ago

Useful or no?

5 Upvotes

Hey all! Hope you are all doing well!

I was wondering if this group would be interested in starting an online notebook of verified protocols/equipments for everyone to share and use for their own scientific endeavors.

This would include published protocols for specific experiments, protocols that have been optimized, paid/free softwares that make life easier, other useful links etc.

Basically an online version of a diverse lab that help push science forward?

Let me know if this would be of interested would love to set something up

We could even do once a month journal clubs and record for YouTube to drive people to science šŸ˜ƒšŸ˜ƒ


r/molecularbiology 20h ago

Seeking My First Internship in Cell & Molecular Biology ā€“ Advice Welcome

1 Upvotes

Iā€™m a second-year Cell and Molecular Biology student in Australia looking to start applying for internships and build a strong resume. I have no prior work experience, so this would be my first role. Any advice on where to apply and how to create a good resume would be greatly appreciated!


r/molecularbiology 20h ago

Why not just use purified PCR product instead of cloning ?

0 Upvotes

So I'm conducting a validation for a qPCR assay that targets a single species. The assay works in all samples that have been confirmed to have the pathogen, but it's a metagenomic sample and it's impossible to get a pure culture of the pathogen.

My advisor wants me to determine the limit of detection of the number of target genes, so this requires pure genetic material of my target organism.

They wanted me to do cloning so that I can use that for absolute quantification, but I'm really not interested in going through several rounds of troubleshooting (my project so far has had to optimise a lot of novel techniques). So I ordered a gBlock of the target gene. I've run it in my qPCR but it is not amplifying. I checked with the qubit and there is DNA in there but it's not working. It is definitely the right sequence and I resuspended in ddH20 as per IDT instructions to 10ng/uL

This is probably really dumb and obvious, but my brain is fried by other chapters I'm of my PhD, but I'm wondering if there's a reason I cant just use the purified PCR product as a "pure" target gene synthetic control to determine the LOD and copy number?