There seems to be a lot of variation in protocols in the time required to dry the beads after the 70% EtOH wash. I've seen from 30 sec at room temp to this video recommending 3-5 minutes in a heat block. I've recently been doing 2-3 minutes at room temp. As mentioned in the video, it will depend on environmental factors (humidity and temperature in the lab) as well as the volume of beads, but I'm wondering if 3-5 minutes in a heat block is really necessary, and may cause too much risk of over-drying. I think the critical point is that the ethanol has all evaporated. If some liquid water remains which causes the beads to appear slightly wet (shiny) that should be no problem, as long as the ethanol is gone, right? Or is getting the beads completely dry specific to SureSelect?
I have issues on certain samples with the beads not fully sticking to the tube wall when in the magnetic rack. This only happens after washing/drying and the beads start to slip down/follow the supernatant down the side of the tube when I'm removing it. I can barely get any volume before I visibly collect beads.
The PEG in the AMPure bead buffer pushes DNA to bind with the carboxyl groups on the Ampure bead surface. The size selection is mainly based on the concentration of PEG, and we use different DNA vs beads ratio to achieve the optimum PEG concentration for the DNA fragment size selection.
AMPure beads are used for the size selection and the post enzymatic reaction purification in NGS library preparation. Although it is technically possible to separate a captured library by centrifuging AMPure bead, it is not a recommended procedure. The magnetic property of the beads have the advantage of obtaining the maximum elution volume from a purified library. Any trace amount of contaminants will have negative impacts in the downstream reactions and quantity/quality assessments. It is recommend to use a magnetic stand when performing the AMPure XT beads purification.
The official Ampure XP Beads protocol (bit.ly/2FUkHLX) mentions volumes down to 5 microliters. I suppose it may depend on what size range you're targeting though.
Thank you. Starting my BCR repertoire profiling journey, and bead clean up is the step I mess up by far. You've helped immensely!
Great video, very helpful for those relatively new to magnetic bead based DNA capture methods.
Nice explanation
Hi, thank you for the video. Is it ok to use any magnet or we need a special type - can you please suggest if yes?
There seems to be a lot of variation in protocols in the time required to dry the beads after the 70% EtOH wash. I've seen from 30 sec at room temp to this video recommending 3-5 minutes in a heat block. I've recently been doing 2-3 minutes at room temp. As mentioned in the video, it will depend on environmental factors (humidity and temperature in the lab) as well as the volume of beads, but I'm wondering if 3-5 minutes in a heat block is really necessary, and may cause too much risk of over-drying. I think the critical point is that the ethanol has all evaporated. If some liquid water remains which causes the beads to appear slightly wet (shiny) that should be no problem, as long as the ethanol is gone, right? Or is getting the beads completely dry specific to SureSelect?
I have issues on certain samples with the beads not fully sticking to the tube wall when in the magnetic rack. This only happens after washing/drying and the beads start to slip down/follow the supernatant down the side of the tube when I'm removing it. I can barely get any volume before I visibly collect beads.
Thank you for the video. Any tips for doing this protocol with a full 96 well plate? I'm worried about the timing of everything.
what happens if you accidentally washed your beads with absolute ethanol? asking for a friend
I'm the friend
What is the ratio of dna and ampure xt bead? thanks
can i use 80% ethanol to wash?
what are the beads binding to exactly? Like is it binding to the barcode? thank you!
The PEG in the AMPure bead buffer pushes DNA to bind with the carboxyl groups on the Ampure bead surface. The size selection is mainly based on the concentration of PEG, and we use different DNA vs beads ratio to achieve the optimum PEG concentration for the DNA fragment size selection.
Hello, it can be done without the magnetic stand?
AMPure beads are used for the size selection and the post enzymatic reaction purification in NGS library preparation.
Although it is technically possible to separate a captured library by centrifuging AMPure bead, it is not a recommended procedure.
The magnetic property of the beads have the advantage of obtaining the maximum elution volume from a purified library.
Any trace amount of contaminants will have negative impacts in the downstream reactions and quantity/quality assessments.
It is recommend to use a magnetic stand when performing the AMPure XT beads purification.
What is the purpose of the beads and ethanol? Thank you!
beads to capture DNA, ethanol to wash and remove contaminants.
hi, can this also be done with low pcr volumes? like 10 microliters?
We've not tried that before, do let us know how that turns out.
I used it for a multiplex pcr, I lost quite a lot(pcr products that were around 110bp were lost entirely)
Let's troubleshoot this further, please submit an inquiry at www.genomics.agilent.com/article.jsp?pageId=3272.
The official Ampure XP Beads protocol (bit.ly/2FUkHLX) mentions volumes down to 5 microliters. I suppose it may depend on what size range you're targeting though.
Nice video guys, too bad your lab coat is torn hahahahahaha!!!!!!!!!!!!!
Hi Ian, are you referring to the side vent next to his pocket? If yes, that is by design for access to the pockets.
Ohhhh l didnt know, it looks torn!!!! Thank you, l have learnt something new today plus the process itself :). Thank you and do have a wonderful day.
Discover new things everyday :)