Electrophoresis Kits

[bigmack]

Just wondering how your set-up has endured, bigmack?

We've used it 4 or 5 times so far .
One tray did develop a slight leak which I think was due to a printing issue .Otherwise they are all working fine .

The dodgy looking Power supplies are still working , but I did manage to squeeze enough from the budget to buy one real one .I hope to be able to replace a few more .

[Marama T]

I am amazed! And now feeling slightly inadequate.

[RosalieL]

Bigmack - I have an appointment on Thursday to talk to my principal (who is also the D&T teacher) to discuss 3D printing one (or more) of these. What info do I need to take in with me to build my case?

[bigmack]

Bigmack - I have an appointment on Thursday to talk to my principal (who is also the D&T teacher) to discuss 3D printing one (or more) of these. What info do I need to take in with me to build my case?

Hi Rosalie
I guess its a matter of Cost verses skills and time .
also how many do you intend to have ?
I ended up buying "proper" Powers supplies to replace the home made ones .
This one was the cheapest and can run 3 chambers .

Power Supply.png

To make the chambers your self , you'll need to download the 3D STL file and print the cases . Then you need a piece of 2-3mm Perspex cut for the bottom .My TAS folk (D&P) had it cut on the laser cutter in about 5 minutes .Then I just needed to glue it in the bottom with silicone .

The electrodes can be as simple as a piece of wire but they tend to do funky stuff so I made the carbon blocks .A bit of effort with a hacksaw and a file , bit not hard at all .

Your Principal may look at it and find $500-700 per box is a lot easier to find than a few of his hours .

I just noticed these are now available . They include a USB powered Power supply .
so if your school is small , a couple of these might be a viable alternative .

Electrophoresis.png

You will also need to get a set of Dyes . I think Southern Bio sell them for around$30 .You will get many goes from them so long as the Kids don't spill or cross contaminate them . You will also need to buy some Micro Pipettes if you don't have any .10uL ones .

I would strongly recommend going Dye Electrophorsis as apposed to Using real DNA samples as these are bloody expensive , have a short shelf life , require expensive Agarose , dying ,washing and take more time to run .Every teacher that used real DNA cried .

[RosalieL]

Bigmack - I have an appointment on Thursday to talk to my principal (who is also the D&T teacher) to discuss 3D printing one (or more) of these. What info do I need to take in with me to build my case?

Hi Rosalie
I guess its a matter of Cost verses skills and time .
also how many do you intend to have ?
I would strongly recommend going Dye Electrophorsis as apposed to Using real DNA samples as these are bloody expensive , have a short shelf life , require expensive Agarose , dying ,washing and take more time to run .Every teacher that used real DNA cried .

Thanks for that! I may just ask to get one of these Bandit ones you've mentioned for starters - I was looking at them yesterday afternoon and wondered if they might be a better (easier) alternative to 3D printing. The other school I worked at used the real DNA and yes, it was expensive, difficult and rarely worked because the teacher who used it was the last-minute type and so everything was usually out of date...

[bigmack]

Fun and games at my new school in QLD .

Been here 2.5 years and they just asked for Electrophoresis kit to be purchased . I found we had one . Its an Advotek one . Has a main tank that can hold up to six trays .
Looked in pretty good nick .

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There was loads of extra Agarose and buffer and a power supply . Couldn’t see why I should bin it and buy a new one .

It comes with some tubes that contain sets of dyes , instructions and enough agarose and buffer to run them . Do these look familiar to anyone ?

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Alas the dyes had all dried up .I searched online but couldn’t find out much . Then I saw it had a Ciderhouse sticker on it .
I gave them a call and they checked and we purchased it 21 years ago in 2005 . OMG

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I asked if they still sold the dye kits but they said no .
The lady on the phone suggested maybe I should try rehydrating them .

So I got out a 1ml hypodermic and pieced each vial lid and injected a few drops of water . A few shakes and sure enough , I had dye that was starting to really colour up .

In the meanwhile , I got an email back from Ciderhouse saying they had found the original dye kits are still available .

No prices at this stage , but this is very exciting .
One thing that looks good about these Advitek dyes is that they all run in the same direction ( start at one end ) whereas the ones I used to use from Southern bio had the start in the middle of the agar and would run in both directions …… OK , but not quite how DNA runs .

Anyway , going to set this up in the prep room on Monday and see if it all works and if my hydrated Dyes work . I’m not feeling confident , but nothing to loose .

[Pm706Narrogin]

We've got our own powders to make dyes so I've never checked out the Southern Biological kits. Just did and what the hell? Why have one run in the opposite direction?

We use xylene cyanol, bromophenol blue, orange g, tartrazine and rose bengal in various combos. Our long-standing lab tech was amazing and managed to wrangle the funding to get our own set of tanks, power supplies, micro-pipettes, tips and the dyes. She also helped the teachers come up with 3 scenarios - muscular dystrophy, paternity, and marine forensics. Rather than running the straight dyes, we make up mixed samples from the dyes that the students get to pipette into their gels and they've then got to interpret the bands according to the scenario.

Feels a bit more interesting than just identifying 2 unknowns that Southern Biological has

[bigmack]

We've got our own powders to make dyes so I've never checked out the Southern Biological kits. Just did and what the hell? Why have one run in the opposite direction?

i know right . It detracts from what the whole simulation is trying to achieve .

We use xylene cyanol, bromophenol blue, orange g, tartrazine and rose bengal in various combos. Our long-standing lab tech was amazing

Thankyou so much . Your old labby sounds like a gem .We had a dozen of dyes and stain powders at my old school and I tried most of them , but only found two that ran the same way , and a couple that ran the other . Some just wouldn’t work . We never had most of what you just listed though

This the way go go forward .

I did make up practice dyes with Cresol green and bromophenol blue with glycerine and water ( similar proportions to Southern Bio uses) but it would start yo grow mold by the next year .

What do you add to your dye powders ? The glycerine is supposed to make them sink i to the well.

[bigmack]

Ouch , this could get expensive .
First cab off the rank .
Xylene cyanol

10g , $144

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[Pm706Narrogin]

Yes we add glycerol to ours and, annoyingly, have those same issues with mould. Ours don't seem consistent with when they grow mould - some are fine for a couple of years, others only last a year. We use our set up roughly 2-3 times a year with Biology and Human Biology so I try to minimise the amounts I make so I'm not chucking away too much cause the powders are bloody expensive.

We've also got silicone moulds for the kids to practice using the micropipettes prior to the real thing but we only use water with blue food dye in it for that. From what I can see, the water gets into the wells in the moulds just fine. I may have to try with the agar to see if the glycerol is really necessary.

I'll try to remember to download our tech notes for our electrophoresis scenarios as it includes the quantities to make up so you can give the students 10μL aliquots. Southern Biological reckon they provide 450μL of each dye in their kits which from memory should be enough to prepare the dye mixtures.

Maybe an affordable compromise could be getting the $50 dye kit from Southern Biological and seeing if you could buy in the tartrazine and rose bengal to make up the other dyes?

Not that I'm thinking of moving from my current school any time soon but my word I'll miss how good this set up is if I go anywhere else

[bigmack]

Thats a good idea PM708.
I have Southern bio ones on the way just in case the hydrated ones don’t work
The rose bengal and tartazine seem go be relatively cheap , circa $20-40 , so could start with them with the Southern bio dyes and and slowly build up the rest .

You can get a lot of runs from the Southern bio ones . Biggest problem was kids knocking them over or cross contaminating them .
To over come the knocking over , I found some big old rubber bungs with holes in them that would hold them safely .

[RosalieL]

I just use food colouring and watered down glycerine stored in specimen jars for the practice gels and they've lasted ages (a couple of years so far).

[bigmack]

I just use food colouring and watered down glycerine stored in specimen jars for the practice gels and they've lasted ages (a couple of years so far).

Good to know Rosalie. Food dye is cheap .But I wonder if they put a preservative in it ?

I rehydrated my 20 year old dried up dyes on Friday . Today I ran it . 20 year old Agarose powder and Buffer .

These are the rehydrated samples after injecting them with distilled water and shaking .

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I setup two trays out of six and tried the dyes from two of the experiment kits .

electrophoresis running.jpg

Alas I forgot to take a pic when it finished .

It took about 20 min but it did work much to my total amazement .Not 100% by any means , but who would have thought you could bodgy up what looked like bin material and it still works .

Anyway , I got a reply back from Ciderhouse and they advised me that although the kits were still available from Denmark , they had explicit instructions to refrigerate immediately , and based on that , they didn't recommend we get them . ....Bugger .

So for now , I have taken PM708 suggestion and ordered rose bengal and tartrazine .The other three are in the Southern bio dye kit.

PM708 , would love to hear how you prepare them .

[Pm706Narrogin]

Annoyingly, our tech notes are too large to attach as separate files so I'll give an overview:

The basic idea is rather than the Southern Biological experiment which simply uses the properties of the dyes to create different bands, if you mix the dyes you can create simulated samples that fit a scenario that the students have to solve. Marine forensics is for when Biology does it and paternity/muscular dystrophy is for when Human Biology does it.

We make up the dye samples and the students are then given them in colour-coded eppendorf tubes. The advantage of this is it eliminates the issue of the students accidentally contaminating the main sample. We have 8 tanks so we work on 8 groups where each group gets 10μl of each sample.
To use Marine Forensics as an example:

The scenario:
A commercial fishing boat has been captured in Australian Antarctic waters. Fisheries officers boarded the boat and discovered several different types of fillets in storage. They suspect some of these to be Antarctic Minke Whale meat but the skipper claims they are tuna. Several samples of the meat are taken to be tested.
Your role: Identify the species from the pieces of meat.

The dyes:

This is where we really engage the brain cause this is a two-step process. The first step is using our individual dyes to make our mixed samples. We're then gonna dispense those mixed samples into eppendorf tubes which the kids will be using in the classroom.

For Marine Forensics, we need to make up 2 mixed samples - one to represent the tuna (we'll call this Dye 1) and one to represent Minke whale (we'll call this Dye 2)

Dye 1 (tuna) is an equal mix of Rose Bengal, Tartrazine and Xylene Cyanol

Dye 2 (Minke whale) is an equal mix of Rose Bengal, Orange G and Bromophenol Blue

The students get 5 samples total in their colour-coded eppendorf tubes:

Yellow: Tuna (known) - Dye 1

Pink: Minke whale (known) - Dye 2

Green: Unknown Fish 1 - Dye 1

Blue: Unknown Fish 2 - Dye 2

Red: Unknown Fish 3 - Dye 2

If you've done the Southern Biological version, the principle is the same - the students are trying to work out what the unknowns are by comparing them to how their known samples run. In this scenario, they should find that Unknown Fish 1 is tuna but Unknown Fish 2 and 3 are illegally caught Minke whale.

The calculations:

So now we work backwards to calculate how much of each dye we need to make.

For each group, they need 2 lots of Dye 1 (or 20μl) and 3 lots of Dye 2 (or 30μl).

Which means if I'm doing my full set up with 8 groups, I'm going 8 groups x 20μl of Dye 1 which gives me 160μl. To give yourself some spare (and make it a nice multiple of 3), up it to 180μl and pipette 60μl each of Rose Bengal, Tartrazine and Xylene Cyanol.

Do the same thing for Dye 2. 8 groups x 30μl or 240μl. That's a nice round number so you'll need minimum 80μl each of Rose Bengal, Orange G and Bromophenol Blue (but again preferably up it for spares).

If those Southern Biological vials give you 450μl of the expensive dyes, you'll have more than enough to cover that set up.

I know that's a wall of text so I've attached the table from our tech notes for the "cliffnotes" version of the recipe/set up

[Pm706Narrogin]

Paternity

The scenario: A fertility clinic has recently been on the news, having been accused of mishandling samples. A couple who used the clinic have been informed one of the mishandled samples may have been used in their treatment. The couple are now seeking to verify the paternity of their child before deciding if they need to pursue legal action.

Your role: using gel electrophoresis you are required to identify the father of the child from 3 possible fathers.

The dyes:

Just like the Marine Forensics, we're going to mix our stock dyes first and then dispense the mixed dyes into the student samples. For this one, we need 4 dye mixes.

Dye 1: Orange G and Tartrazine

Dye 2: Bromophenol Blue and Orange G

Dye 3: Rose Bengal and Tartrazine

Dye 4: Bromophenol Blue and Xylene Cyanol

The samples:

Again we use colour-coded eppendorf tubes but you can designate them however you want.

Yellow: Mother - Dye 1

Pink: Child - Dye 2

Green: Possible Father 1 - Dye 3

Blue: Possible Father 2 - Dye 4

Red: Possible Father 3 - Dye 3

So for this one, if we're working on 8 groups at 10μl per group, I'd need 80μl of Dye 1, 2 and 4. To allow for spare, make 100μl of each dye by mixing 50μl of your stock dyes. Double it for Dye 3 (which, coincidentally, are the cheaper ones not in the Southern Bio kit).

The result:

In this scenario, the child's sample is made of Bromophenol Blue and Orange G. He's inherited the "Orange G" from his mother and since Possible Father 2 is the only sample with Bromophenol Blue, the child must have inherited it from this father.

As a bonus, Possible Father 1 and 3 being made from identical dyes, the kids can also work out that these samples came from identical twins.

[Pm706Narrogin]

Muscular dystrophy:

This one is really good for Human Bio classes who have a good handle on inheritance. The results from this one requires a bit of thought to figure out.

The scenario: A family consisting of a father, mother and four children have had some devastating news. One of the children has recently been diagnosed with Muscular Dystrophy and the parents are now worried that the other children may pass the disease onto future generations.

Your role: Using gel electrophoresis, you are required to identify which child has the disease and if any of the other children are carriers.

The dyes:

You know the process. Use your stock dye to make up mixed samples and dispense to the students. This one also requires 4 dyes.

Dye 1: Rose Bengal and Tartrazine

Dye 2: Rose Bengal and Bromophenol Blue

Dye 3: Rose Bengal

Dye 4: Tartrazine and Bromophenol Blue

The samples:

Orange: Father - Dye 1

Purple: Mother - Dye 2

Green: Child 1 - Dye 2

Blue: Child 2 - Dye 3

Red: Child 3 - Dye 4

Yellow: Child 4 - Dye 1

So again if I'm working on 8 groups, I need minimum 160μl of Dye 1 and Dye 2 and minimum 80μl of Dye 3 and Dye 4. As before, make up extra but again you should have no issue getting what you need out of the Southern Biological kits. The more I look into this, the more I'm feeling these dye pairs were very deliberately made up to use more of the cheaper Rose Bengal and Tartrazine.

The results:

This one I've found is a bit tricky to interpret so I've attached the dye recipe/answer key below

[bigmack]

This is awesome info PM706

Just another question , well two .
how much dye powder do you use (what is your dye recipe )? and do you make it up fresh each time ?

For example , the southern bio ones say 0.2% water/glycerol (80/20)
my take on that is 0.2% w/v of powder and the liquid is 80% water and 20% glycerol.
(But then again, I could be completely wrong about that .)

so if you have to make up 180uL , 0.2% w/v would be 0.36mg .My most sensitive scales only go down to 1mg .So how do you weigh out the powder or do you make up a larger amount and store it in the fridge so it won't go moldy .

[Pm706Narrogin]

We make up 100mL quantities and store in the fridge. Mould is the only issue so when we need to, we'll make up the 100mL of whichever stock dye we need (e.g. Tartrazine) and that is right to go for as long as it lasts without growing mould. All things going well you can get a good 6-12 months out of them in the fridge so one batch tends to do at least a year of use.

Yeah we follow the Southern Bio recipe to make up the stock dyes. So 0.2% w/v in an 80-20 ratio of water and glycerol but made up to 100mL portions. We then pipette from these stocks when we follow the process for making up the student dyes.

You could even afford to go lower than 100mL to reduce the amount of wastage further. Whatever you find is the right compromise between minimising waste and being something you can actually measure

[bigmack]

Thankyou so much.

[bigmack]

My Rose Bengal , tartazine and orange G turned up .

Beware the rose bengal …… should have worn gloves …. A little goes alooooooong way

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