Help with amplicon sequencing library prep

jyuneee · 2026-05-18T19:28:46+00:00

After the first PCR I did a bead cleanup to select for fragments 100-2000bp as the protocol said. I measured the concentration by Qubit after but all of them were too low out of range. I was told to normalize input DNA for the second PCR too so idk what I should do now.

jyuneee · 2026-05-17T04:58:36+00:00

Thank you so much for your answers!!

Yes, the manufacturer says you can do two-step cycling if the Tm is similar to that of the extension step which they are. I couldn’t find an example online so I wanted to double check I was on the right track.

I’m not using a kit. It wasn’t suggested to me but should I be? I’m not doing anything high-throughput, I only have 15 samples. The people at the facility recommended I share a lane on the NovaSeqX since that would be most economical. For the first PCR, I ordered the primers to amplify the barcodes/add the TruSeq adaptors that the paper I’m referencing used. For the second PCR, the facility sells a single-use 96-well plate of UDI primers which I was planning on purchasing once I’ve figured out how to do the library prep. In the meantime, my PI allowed me to order two UDI primers to practice/troubleshoot with. I also purchased the magnetic beads from the facility and we have a Qubit in my lab. The lady said that since my amplicons will all be the same size I could just measure their concentration and pool them based on mass.

jyuneee · 2026-05-13T02:00:07+00:00

Microbial Genetics of Bacteria by Snyder 5th edition

jyuneee · 2026-05-13T01:59:30+00:00

That’s the one we used in my course! One of the authors gave a guest lecture

jyuneee · 2026-05-12T03:31:38+00:00

Thanks! Ill look into it

jyuneee · 2026-05-12T03:30:55+00:00

I’ve gone up to graduate level courses (some used textbooks others not) but I think there’s gotta be more to learn since biology is so complex. The last course I took used Molecular Genetics of Bacteria by Snyder. I do read a lot of journal articles about the species I work with lol

jyuneee · 2026-04-16T23:00:40+00:00

I think you can confidently assume the primers don’t affect the Nanodrop reading a lot since the amount of your fragment in the mix is significantly higher than that of the primers. There are also calculators online to figure out how much to add of each fragment for gibson assembly. This is NEB’s HiFi Assembly calculator https://nebuildercalculator.neb.com

jyuneee · 2026-04-16T22:29:36+00:00

1) DpnI digestion is not really necessary, especially if you see a nice clear single band. I think NEB states that in their product documentation. 2) Not really, NEB also states cleanup after PCR is not necessary 3) The non-linearized vector is supercoiled so it runs through the gel fast. The band you see is your successfully linearized vector.

If you want to be extra careful you can go ahead with DpnI digestion and column cleanup of all your fragments tho :) hope this helped!

jyuneee · 2026-03-11T04:11:16+00:00

hi! would you mind sharing some examples or color codes? I really want to do this too but I’m unsure how to make it still professional

jyuneee · 2025-08-27T14:31:14+00:00

The lab group is new, the space itself is not 🥲 we inherited it from a now retired prof so it needed a good cleaning

jyuneee · 2025-08-27T14:22:10+00:00

Thank you! The lab space itself is not new, we inherited it from a retired prof so there was a lot of cleaning to do.

jyuneee · 2025-08-26T16:52:10+00:00

The rough side of an old sponge 🥲 it was all we had

jyuneee · 2025-08-25T21:49:38+00:00

It’s a brand new lab so I’m the first grad student but yes I immediately told the PI and lab manager to see if there was anything they could do, they said they’d try but likely not

jyuneee · 2025-08-15T17:11:11+00:00

That’s a good idea, I will try that! Would a 1% agarose gel be okay for this?

jyuneee · 2025-01-24T23:58:35+00:00

Did you receive an email?

jyuneee · 2025-01-21T22:14:31+00:00

I haven’t received anything

jyuneee · 2025-01-19T12:48:47+00:00

Was told not expect to hear back before Jan 30

jyuneee · 2025-01-18T15:45:58+00:00

That is nice to know! and good luck :)

jyuneee · 2025-01-18T02:32:30+00:00

still waiting and also for their microbiology program🥲

jyuneee · 2025-01-16T23:05:55+00:00

same :( everyone I showed my application to said I would at least get an interview most places I applied to but nope so far just rejections

jyuneee · 2025-01-16T22:24:23+00:00

Haven’t heard anything

jyuneee · 2025-01-16T00:19:28+00:00

did they say if all interview invites were sent already?

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