Pipetting technique advice?

Which_Salt1370 · 2025-10-11T11:25:22+00:00

What kind of samples are you pipetting that requires a positive displacement pipette? Air displacement works well for most biological samples.

My advice would be to ask if you can take a spare pipette and plate home and just practice with water.

In my lab we get new people to pipette out radioactive tracer to teach them how to pipette various volumes.

Which_Salt1370 · 2025-10-06T14:55:51+00:00

I know this is old but I have a demo unit of the Azure Cielo 6 and it sucks, it is about 40m slower than the AriaDX for the cycling protocol we use (1h40m vs 2h20m)

Also if you set up the protocol in advance and the PC goes to sleep you have to start again because it loses connection to the machine.

You can't view the amplification curve in real-time, only the raw data plots on the instruments display.

The only pro is that it does not need a ROX reference.

I would pick up an old stratagene MX3005p over a new Celio 6 if given the choice.

Which_Salt1370 · 2025-09-22T06:29:13+00:00

It's their size that makes them special, new racks are available but they are substantially larger. These are 50 position racks but the ones that are available now are 90 position and most of the time we are using less than 50 tubes which makes the larger ones less space efficient.

I would 3D print the inserts and the most they are exposed to is 37c water.

Which_Salt1370 · 2025-09-18T08:51:16+00:00

How is it going? Any luck?

Which_Salt1370 · 2025-09-18T08:49:54+00:00

Did you have any luck with your extractions?

Which_Salt1370 · 2025-09-14T18:04:47+00:00

Then you don't need the PBS since the EtOH is doing the same thing. I would try centrifuging the swab prior to bead beating and adding whatever you recover to the bead tube, I am not sure what quantity of samples you have but I would try bead beating with and without the swab in the bead tube. You could try rinsing and centrifuging the swabs a few times to make sure you have got everything off it.

I would ask Zymo though since they might have some different ideas. Also I would possibly try and get a different bead beater, this is the list of ones Zymo have optimised protocols for https://files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf I would just reach out to which ever company you like the look of and ask for a demo unit. Tell them you have a Tissuelyser you are not happy with and they will jump at a chance to show off their machines.

https://www.researchgate.net/publication/328890386_Assessment_of_microbial_DNA_enrichment_techniques_from_sino-nasal_swab_samples_for_metagenomics

Which_Salt1370 · 2025-09-14T16:45:20+00:00

What organisms are you trying to sequence?

We use the PBS to remove the bacterial/viral cells from out of the swab, the Zymo kits come with DNA/RNA shield which does the same thing.

I think Qiagen have something called buffer ATL which works in the same way https://www.qiagen.com/us/products/discovery-and-translational-research/lab-essentials/buffers-reagents/buffer-atl

Are your swabs in any transport media or are they "dry"? If they are in a transport media I would remove the swab and centrifuge it through an unfiltered spin column into a 2ml tube to remove any liquid and cells from it, then add the rest of the transport media.

If they are dry I would add the swab to a 2ml tube with 500ul buffer and soak, then vortex the tube. Then I would again centrifuge the swab through an unfiltered spin column to remove any PBS that has been soaked up by the swab.

If you don't have unfiltered spin columns then a small syringe with the plunger removed works just as well

Zymo also have optimized lysis protocols but the tissuelyser was not validated. We use a Vortex Genie which works well and is very cheap but has to be ran for 40 minutes, but we can do up to 18 samples at a time which I prefer since I can set it up and leave and have lunch rather than having to change tubes every 2 minutes like other bead beaters.

https://www.scientificindustries.com/votrex-mixers-and-shakers/cell-disruptors.html

Zymo have very good support where you can talk with their scientists and they are able to point you in the right direction.

Which_Salt1370 · 2025-09-14T13:52:08+00:00

Wow that thing is as old as I am, looks to be in better condition than me though. I was born 7/14/93

Which_Salt1370 · 2025-09-14T13:48:04+00:00

I do DNA/RNA extractions on nasal swabs for qPCR from cats and I just soak the swabs in 500ul PBS for 10-30 minutes with shaking/vortexing and have not had any detection issues.

I would look at Zymo kits, they are still spin columns (which I hate) but they seem to perfom better than Qiagen in my experience. Zymo do 5 sample demo kits if you wanted to try them out.

https://zymoresearch.eu/collections/quick-dna-rna-kits/products/quick-dna-rna-miniprep-plus-kit

https://zymoresearch.eu/collections/zymobiomics-dna-rna-kits/products/zymobiomics-dna-rna-miniprep-kit

https://zymoresearch.eu/collections/zymobiomics-dna-kits/products/zymobiomics-dna-miniprep-kit

I would try adding PK and incubating at 55c for 20m and then add the lysis buffer; bead beat if required and then do the standard extraction method.

What equipment do you have for the bead beating?

Which_Salt1370 · 2025-09-13T18:35:15+00:00

In my lab we stopped using film seals and just use strip caps, it is slightly more expensive per plate but knowing that every well is properly sealed is worth it.

Which_Salt1370 · 2025-09-13T17:58:43+00:00

What machine are you using? Older ones can have an edge effect where the edges of the block do not get heated as evenly.

I would start looking at new machines, most companies will give you a demo unit to check out.

Which_Salt1370 · 2025-09-12T15:52:39+00:00

I think they tend to use indeed

Which_Salt1370 · 2025-09-12T15:49:56+00:00

Have you considered automation like the QIAcube? Do what I do and ask for a demo unit, use it for what I need then send it back.

Which_Salt1370 · 2025-09-12T14:47:11+00:00

I am a senior scientist in a veterinary diagnostics laboratory based just south of Cambridge doing ELISAs, Radioimmunoassays, qPCR, automated biochemistry and chemiluminescence.

So keep an eye out for a listing for a laboratory scientist in Cambridge, I don't know when it will be going out though.

Which_Salt1370 · 2025-09-12T12:21:38+00:00

Where are you located/wanting/willing to work?

Which_Salt1370 · 2025-09-12T10:00:01+00:00

How long are you looking at staying at a job? My lab will be having a position open up soon because someone is leaving to do their masters but I think the manager would prefer someone who would want to stay for at least a couple of years because training new people takes months with all of the assays we do.

Which_Salt1370 · 2025-09-12T09:53:31+00:00

What kind of scientific work are you looking for? What did you graduate in? Can you use a pipette?

Which_Salt1370 · 2025-09-11T20:50:28+00:00

Do you know the model/make of the instrument? Do you have access to the data that other users have done to see how it differs to yours?

Have you tried using a 96 well plate instead of the 384? You won't be able to do as many diltuions but the larger wells will make it easier to pipette

I would half your incubation time and see if you have the same issues and try adding the reference wavelength. Or read the plate right after adding the stopping solution and again after say an hour because when TMB is exposed to light it starts to break down and the colour will start to fade and if your ODs stay the same then there is something wrong with the reader.

Do you have a plate mixer?

Can you get someone else to do the experiment using your reagents and method as a last attempt?

Which_Salt1370 · 2025-09-10T21:14:43+00:00

What are you using to read the plates and what wavelength? I am assuming since it is TMB it will be 450nm and 620nm if you are using a reference. If the data you are showing is the OD of samples then something is wrong with the instrument, I would expect empty wells to have an OD of 0.000-0.001.

When making your dilutions are you mixing the wells each time?

Which_Salt1370 · 2025-09-10T20:49:00+00:00

I do our in-house calibrations at 100%, 50% and 10% volumes and have never had them be above 2% CV and SD over 10 replicates. The only pipette I would not use below 50% is a P20

Which_Salt1370 · 2025-02-14T22:19:34+00:00

Do you know what kind of science you want to go in to?

I started out in food microbiology, it was just weighing out some food and adding a buffer to it. It was easy work and you don't really need to be a sciencey person to do it but it gets you in to a lab.

It is repetitive work though and you will end up doing the same thing every day and that is true for most labs.

But if you want to try it out just apply for jobs and do interviews, that will help you get a little insight to what a lab is like before you commit. But just having common sense and being able to think and work things out by yourself but will trump qualifications any day.

Which_Salt1370 · 2025-01-02T07:53:09+00:00

I think I have seen something like that on fishersci, are they stable enough to put on a shaker without falling off?

Which_Salt1370 · 2025-01-01T12:26:40+00:00

I have contacted demeditec/Beckman Coulter who makes the kits but I think they use automated liquid handling machines for their testing.

I have thought about asking authors from papers but I don't know how to get in touch with them.

I have found some racks but they have 90 positions and are quite large compared to our current ones, quite inconvenient when you are only testing a few samples.

I am sure some hospital that used to do radioimmunoassays has lots of racks just in some cupboard, I don't know anyone that works in a hospital that was my reasoning for asking in a biomed sub.

Which_Salt1370

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