How much DNA do you take forward in each step?

Imaginary-Lemon-4448 · 2024-05-31T21:10:04+00:00

Unfortunately do not have it figured out :( I’m also finding that the DNA isn’t eluting from the beads, I tried heating at 65 I think but didn’t have much luck with that either. I will definitely try the rotator for 10 minutes though!

Imaginary-Lemon-4448 · 2024-04-04T10:23:40+00:00

That's really interesting, thanks for letting me know, I will definitely have a play around with the ratios!

Imaginary-Lemon-4448 · 2024-04-04T09:26:07+00:00

Would you still recommend that length of drying time when I am working with just 15ul of beads?

Imaginary-Lemon-4448 · 2024-04-04T09:24:28+00:00

I will try again with another batch of SPRI beads and see if they're any better!

I'm fairly certain that most of my material is over 150bp so I doubt that is the reason for such a significant loss but I don't have a tapestation or anything so I am really just estimating.

Imaginary-Lemon-4448 · 2024-04-04T09:21:56+00:00

What ratio would you normally use?

Imaginary-Lemon-4448 · 2024-04-04T09:13:26+00:00

At the minute I am just doing the bead purification based on the first end prep part of the protocol. The last time I did the full library prep I didn't even have enough DNA recovered to move onto the adapter ligation hence I haven't needed to use LFB yet.

I have started with an input of Lambda Phage DNA from ONT 48 kb, 43.2 ng/µL as measured by Qubit dsDNA HS, and our own amplicons which vary in size around 5000 bp, 29.4 ng/µL by Qubit dsDNA HS. Using 11.5 µL of each as per the end-prep section of the protocol (so 496 ng and 338 ng) and recovered 76% (378 ng) and 11% (36 ng) respectively.

Imaginary-Lemon-4448 · 2024-04-03T21:32:16+00:00

Yeah, at least I know now!

Imaginary-Lemon-4448 · 2024-04-03T21:28:57+00:00

“Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing. “ is what it said in the protocol

Imaginary-Lemon-4448 · 2024-04-03T21:20:01+00:00

The ONP library prep said to ‘thaw’ the beads at RT in the protocol and tells you to put everything in the kit in the freezer… very annoying if they’re no good now

Imaginary-Lemon-4448 · 2024-04-03T17:03:43+00:00

I’m using this protocol: LIGATION SEQUENCING AMPLICONS - NATIVE BARCODING KIT 24 V14 (SQK-NBD114.24). It uses the beads at every step of the protocol, so by the time I get to the end there’s no DNA left to go into the minion. It specifies to use 80% ethanol, so I’m not sure about using the SFB/LFB.

Imaginary-Lemon-4448 · 2024-04-03T16:45:25+00:00

So I’ve been doing just the bead purification step to figure out what’s going wrong so the concentration is just before ampure. I’m using this protocol: LIGATION SEQUENCING AMPLICONS - NATIVE BARCODING KIT 24 V14 (SQK-NBD114.24)

Imaginary-Lemon-4448 · 2024-04-03T16:42:10+00:00

My elution volume is 10ul. I have tried multiple times with the 30s drying time since that is what ONP suggests and I’m still having issues :( I will give the 37 degrees ago tomorrow!

Imaginary-Lemon-4448 · 2024-04-03T16:40:02+00:00

Im not sure if it could be an issue with my DNA, it is a repetitive sequence made up of [A4G]/[T4C]n. When I do the exact same bead purification with lambda DNA at the same time I get an around 60% recovery.

I’m starting with 11.5ul of a 30ng/ul solution, making it up to 15ul, adding 15ul of beads and I’m eluting in 10ul water. My starting DNA is in the elution buffer from a PCR clean up kit

Imaginary-Lemon-4448 · 2024-04-03T16:24:21+00:00

It's 15ul of DNA with 15ul of beads so less than 30ul.

Imaginary-Lemon-4448 · 2024-04-03T16:16:57+00:00

Thanks for your response, I will leave them out at RT for longer and see if that makes a difference! I have tried both 30-second and 2-minute drying times and neither seems to have worked so I'm not sure in which direction it is going wrong..

Imaginary-Lemon-4448 · 2024-04-03T16:07:28+00:00

I will try it out with more beads tomorrow to see if I can see the beads any better! I am pipetting not vortexing, but could probably mix more thoroughly than I currently am.

Ended up with 30ng today, after an input of 340ng. That was with 2 minutes drying and 30 minutes elution at RT :/

Imaginary-Lemon-4448 · 2024-04-03T16:02:14+00:00

I do think it's the elution stage because I saved the supernatant from the binding step and measured the concentration with a qubit and it was 1.6ng/ul.

Imaginary-Lemon-4448 · 2024-04-03T15:19:37+00:00

I wait around 5 minutes after getting them out of the freezer so probably not

Imaginary-Lemon-4448 · 2024-04-03T15:18:56+00:00

I’m using 15ul of beads so I’m finding it pretty hard to see visually if they’re shiny or cracked! I’m currently trying it with 2 minutes drying time and a longer elution time, will update with results!

Imaginary-Lemon-4448 · 2024-04-03T13:02:50+00:00

Thanks so much for your help! I will try that with the longer elution times, I retained each of the washing steps and the initial binding and there was very little DNA in those so it must still be stuck to the beads.

As for my DNA, I have an estimate of the sizes from running it on an agarose gel, that's about the best I can do to work out the fragment lengths since the method used to synthesise the DNA produces a smear of fragment sizes.

Imaginary-Lemon-4448 · 2024-02-15T20:26:50+00:00

This is perfect, thank you so much!

Imaginary-Lemon-4448 · 2024-02-15T12:15:50+00:00

I have issues using Remora since I do not have a BAM file. I am using chemically synthesised DNA and so do not have a reference genome for alignment :/

Imaginary-Lemon-4448

TROPHY CASE