Technical Tuesday: Economical Electrophoresis

Gel Electrophoresis is a method to spatially separate DNA,  proteins or macromolecules in general by their weight (or charge). Depending on your field of research you’ve either never heard of this, or they are ubiquitously referred to as “gels” and they are a part of your daily life.

The authors of this blog work in a gel-philic field.

This Technical Tuesday we are talking about making this process less draining on your finances, whether you are running one or one-thousand gels.

DNA Gels

Gels run to separate DNA are usually agarose gels (0.7-2%). Commercially available pre-cast gels run $10-$20 each. In contrast you can buy enough reagents to make 300 gels for ~$800–which bumps the cost down to around $2.50/gel. These agarose gels are easy to pour because the agarose polymerization is a temperature based transition.

You will also need a running buffer (TAE or TBE). A 50X stock solution of TAE will cost between $50-100.  You can make the buffers yourself  without too much effort, and if you are really committed to saving, some folks say that you can reuse them.

Note that Ethidium Bromide (the most commonly used stain in DNA agarose gels) is perhaps not as dangerous as the signs in your lab make it out to be. There is very little reason to splurge for the more expensive “non-mutagenic” stains.

If your lab is running dozens of gels (or more) each week, you should pour your own–addgene has a good tutorial which any undergraduate could follow.  If you anticipate only running a few gels in your career, it might be cheaper to buy things pre-packaged because the bulk reagents are a hefty upfront cost.

Protein Gels

Similar to DNA gels, it is always cheaper to pour your own gels for protein electrophoresis–pre-packaged gels still run ~$10 a pop . To figure out what kind of gel you need to use we recommend this resource from Thermo Fisher. Sometimes you can get away with agarose gels for very heavy proteins, but usually protein gels are polyacrylamide.

Polyacrylamide gels are harder to pour than agarose gels because the polymerization is chemical rather than physical. Plus, acrylamide is a known neurotoxin so safety is a concern. There are lots of resources out there that describe how to pour your own. Our favorite was proposed by Alexander Hwang, Paris Grey, Katrina Cuddy and David Oppenheimer in this paper.

They’ve given a detailed description of how to retrofit Invitrogen Nupage Novex minigel cassettes for reuse with little more than epoxy. The paper even includes this video instructional:

Gel Boxes

No matter how you are making your gels, even if it’s just two glass plates, some packing tape, a few binder clips, a HDPE comb and your polymer, your going to need a Gel Electrophoresis Chamber. At $500 these don’t cost much, but then again they are just plastic boxes. So why not make your own?

Check out this fantastically detailed tutorial on how to build a gel electrophoresis chamber, put together by Rob Wheeler, and Dr. Louisa A. Stark.

If you want something a little more compact, here’s a tutorial complete with videos on how to make a smaller gel electrophoresis system.

Picture of Gel Electrophoresis System (mini)

Power Supplies

If you want to run your gels, you are going to need a power supply to apply a voltage difference across the gel. Go through this quick instructional on how to build your own power supply to make a compact and beautiful little power supply.

Picture of Gel Electrophoresis Power Supply

Imaging

So you can get really expensive and fancy machines to take pictures of your gels. If you are not too picky about image quality, you can use your smart phone and some good lighting to get decent pictures. Looking for higher quality photos? You can build your own gel imaging box with a  nice digital camera for less than $1000. And even the commercially available boxes are getting cheaper; here’s the imaging box (without the camera) that one of our contributors uses that cost them just $275. Although it does seem like you are just paying for a box with a light at the bottom. And finally here’s a great guide to help you figure out what kind of system to get to fit your needs.

Don’t want to do it yourself but still want to keep the cost reasonable?

Minipcr sells a Electrophoresis Chamber complete with power supply and built-in blue light transilluminator for just $350. The box is translucent so you can take pictures of your gel with a phone as it runs.

Thanks to Dr. John Berezney for pointing out the resources on DIY gel boxes.

Thanks to James Sheehy for some of the information in this post.

Technical Tuesday: Costs of Cooling

It’s Technical Tuesday! This is a rough one, and we hope people will reach out to us and give us additional advice which we will include here. Today we are talking about the costs involved in keeping things cold, and how to mitigate them.

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We’ll start with the not very  cold, and get colder. But first, this:

Chests, Uprights and Best Practices

In your house you have an upright refrigerator, but when you use a cooler, you are using a chest configuration. Chests take advantage of the fact that cold air sinks and hot air rises to maintain their temperature better while open. This is a huge advantage, with an upright -80 C, getting materials costs 2-4 degrees. With a chest, it’s often just 1 degree. Chests also tend to be fail safe, in that it’s hard to leave a chest open by accident. I have literally lost thousands of dollars in ruined chemicals due to people not properly closing freezers.
BUT! They are difficult to clear of ice which falls to the bottom. They tend to be more cluttered, as items can fall into hard to reach cracks. Perhaps most importantly, they take up more space than uprights.
Whatever configuration, the smaller the fridge, the larger the heat flux due to door openings. Small freezers under desks tend to not be as temperature stable as their larger brethren.
For location, think about heat rejection from the condenser, those are the coils on the back of your fridge. This is where your fridge rejects heat into the environment. The warmer the air around those coils get, the lower the energy efficiency and performance of your fridge. So try to not have the back of the fridge in a corner or exposed to the sun.

Refrigerators (273 K / 4 C)

NIST has a nice powerpoint summarizing best practices for using refrigerators. Commercial refrigerators with no built in freezer are great and we love refrigerators with glass sliding doors, as they are easy to use and promote organization.. You’ll want to avoid small commercial refrigerators.

NIST also has suggestions on where you should be putting your chemicals in the fridge: you don’t want to put anything degradable on the top shelf. You can click on these pictures to make them bigger if you don’t want to download the NIST powerpoint.

You’ll need a temperature probe. Like the TIs SensorTag or the WirelessTag, which will both log data for you and can be set up to email you if temperatures drop, or you can opt for something that will just beep loudly when the temperature goes out of range. In my experience the beeper gets shut off by people in the lab who think the beeping is an annoyance.

If you want to pay less for electricity, consider retrofitting a freezer that has better insulation with a Johnson Freezer Temperature Controller. You can read about building a “chest” style fridge out of a freezer using such a temperature controller in this really cool tutorial.

freezer fridge

Ice and Ice Makers

You want a Flake Ice Maker, which luckily is used in restaurants and hotels. I recommend the smallest Hishizaki brand ice maker you can find. When it comes to working with the ice, there’s no real reason to buy an ice bucket. Small Styrofoam boxes work great and are probably already abundant in your lab.

Image result for styrofoam ice box lab

Freezers  (253 K / -20 C)

Again, it’s perfectly fine to go commercial here and much cheaper.  Again, get something to monitor the temperature, preferably something that sends out emails when the temperature falls too low as discussed in the refrigerator section.

Whether it’s an upright or a chest, get it commercially, and get racks for your freezer.

Image result for freezer racks for 15/50ml conicals

As a default, get a manual defrost freezer and be ready to empty it out every once in awhile. If you do get a auto defrost freezer, be careful and see if you can get one with cycles you can control.

Dry Ice

You can get dry ice for about $2 per kilogram. With reasonable insulation, you can expect to lose 2-3 kilograms every day. While dry ice is mostly used for shipping, if you want to keep just a few things at -80 C for just a couple weeks, it might not be worth the investment to get a -80 C. At $42 a week, you would probably be able to buy a year or two of dry ice before it started to match the cost of the cheapest -80 freezers. Of course you wouldn’t get much volume of sample storage that way.

Freezers  (193 K / -80 C)

Not Thermo Fisher.

Panasonic/Sanyo is highly recommended, though not particularly cheap. I cannot stress enough however, that your going to lose a lot more money and time on dealing with -80 Freezer problems. This simply isn’t a good place to skimp.

If the freezer doesn’t come with an auto-call or email system in place, you’ll want to rig one up again. Just make sure your hardware can withstand the cold.

Cryogenic Freezers & Liquid Nitrogen (77 K / -196 C)

Liquid nitrogen will cost around $1 / liter, often less. In a 6 liter dewar, you can expect half a liter to boil off each day. In theory, if for some reason you don’t have access to it, you can just produce your own liquid nitrogen.

A dewar meant for holding samples in liquid nitrogen is called a cryogenic freezer. Cells or Cryo-EM samples often need a cryogenic freezer. Collectively here at LabOnTheCheap we’ve only worked with two cryogenic freezers, none of us remember the brand names, one was an auto-fill, the other was a manual fill, and both sucked.

Advice? Suggestions? Used a cheap cryogenic freezer you can recommend? Email us at LabOnTheCheap@gmail.com

Thanks to Dr. Bryan Kaye for information regarding refrigeration.

 

 

 

Technical Tuesday: Dealing with Data

It’s Technical Tuesday! Today we are diving into the controversial topic of paying for data storage, and of course since the two go hand-in-hand: backup solutions. There is no need to take any of this as gospel. We are presenting one way of many of going about this.

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Start with Cloud Storage:

The cheapest solution to data storage is Cloud Storage, which to an extent is free.

As an example Google Drive offers 15 GB to any google account for free. You can set it up to automatically sync with multiple drives on multiple computers, and it will not just back up your data, but even keep track of revisions.

If you are associated with a large company or any university, almost everyone with an .edu email address can get unlimited storage on Google Drive.

If you don’t have such a hookup, no worries. You can get drive for work, which runs $10 a month for unlimited storage or  you might be eligible for G-Suite for education, which will get you that unlimited storage for free.

If none of these situations fit you, you can always get a personal license. Amazon only costs $11/year for 100 GB , or $60/year for 1 TB. 

Google Drive is just an example. Your university or place of work might have unlimited storage on DropBox, Amazon, Microsoft OneDrive or a myriad of other sources.

A final note, if your pieces of data are very small or very big compared to 500 MB consider piecing them up into 500 MB chunks in an archive format such as ZIP or RAR. Cloud services can have trouble with handling tens of thousands of files at a time or files on the order of a TB. This also gives a benefit of also reducing the size of certain files (for instance my photometry data is reduced by a factor of 2 using ZIP).

When to augment your cloud storage? 

It depends on your upload speed. Here’s a little test to see how fast your internet is.

Annoyingly upload speeds are reported in megabits (Mb) instead of megabytes (MB). 8 Mb/s is one MB/s. So divide the number you get here by 8.

Optimistically, your internet when plugged in by Ethernet is  between 1-10 MB/s . This means that somewhere between 50-500 GB of data a day you are going to have to find additional storage solutions.

Before You Start: Try IT

If you have such a resource, contact IT about getting a faster internet connection. If you are paying for your own internet, it might be worth considering an upgrade.

If you are at a larger institution, ask IT about Research Cluster Data Storage. As an example of what you are looking for, check out this page of Harvard Medical School for a cluster they run. Institutional solutions will often have much higher upload/download speeds on their intranet, or private network.

Augment Cloud Storage with A  Storage Server

If you find you are limited by your upload speed, consider augmenting your free cloud storage by moving some of your data to a local storage server.

If you don’t have enough confidence to build your own server, you can buy one. Consider purchasing last-gen servers that businesses are getting rid of on eBay or Craigslist. Check out this page which has what to look for as well as things to watch out for. If you want to buy something new but provide your own drives consider 45 Drives.

If you want to build your own server on a budget (~$1000 not including hard drives), here’s a possible outline for you

  1. Processor: A low-tier Xeon Processor with integrated graphics along the lines of of an E3-1225 V5
  2. Memory:  ~8GB of ECC RAM
  3. Motherboard: Choose a board to match your processor socket and supports ECC RAM. 
  4. SATA Controller: Often, you can’t physically plug in as many hard drives as you want. You need to get an expansion card. You can get a card like this and then use 4 in 1 breakout cables to plug up to 8 HDDs into it.
  5. Internal Power Supply: Just make sure you have enough connectors for as many HDDs as you want to run, or additional cables have a  place to plug in if necessary.
  6. UPS: To protect the server from sudden power outages, consider a Uninterruptible Power Supply.
  7. Case:  You could go for a reasonable dedicated server case that is typically in a rack mount form factor. Or you could buy something enormous.

If you feel like some hardcore DIY, that 600 TB setup is open source by BackBlaze.

In the case of a dedicated server you want Western Digital Red branded hard disk drives. Right now 4 TB ($125) and 8 TB ($260) drives are particularly cheap per GB, and both come with 3 year warranties.

Finally you’ll need a server operating system (OS). We recommend unRAID. For the ultra budget conscious consumer, you can often get a Windows Server license from your local IT department. Or tackle your problem with a free Linux distributions (Mint, Ubuntu, Arch, CentOS, etc.). These take some configuration in order to set up a filesharing server (making a ZFS pool, etc.), but can be fully capable. Finally, there are alternative NAS OS that are free (FreeNAS, and Xpenology for starters).

The benefit to unRAID is that it only requires two backup drives for your whole server (to a point). Instead of me fumbling about how parity backups work, just read this. For those of us who grew up with RAID 1, this is black magic. It also makes it easy to have a speedy set of cache drives that let you quickly upload data which is stored in the slower storage drives over time. unRAID maxes out at 24 storage drives, which with 8 TB drives means 192 TB. If you hit that limit, you can always build another server.

If you want to access a personal server, be it local or hosted by someone else, with the same ease as Google Drive on all your devices, consider installing a platform such as NextCloud on your server.

Augment A Storage Server with Personal Drives

Finally, if internal servers and the cloud simply don’t cut it, try to put the really big files in physical drives.  At 0.75 GB/s (That’s 6 Gb/s, I know confusing right?), you can put a drive into a hot-swap port, transfer data onto it, pull it out and bring it to another computer for back up and analysis.

Image result for WD blue 4tb

For a drive that will be moved around a lot, you want WD Blue. a WD Blue 4 TB drive comes in at just $100 right now.  This includes a 2-year warranty. You’ll pay $50 per TB for the drive and a backup drive.

StarTech.com 5.25in Trayless Hot Swap Mobile Rack for 3.5in Hard Drive - Internal SATA Backplane EnclosureTo move data around, install a Hot Swap SATA port to your desktops. For around $10 each, these will let you easily transfer drives from desktop to desktop.

Cable Matters USB 3.0 SATA HDD/SSD Docking Station with 10TB+ drive support (Thunderbolt 3 & USB-C compatible)Buy $20 Hard Drive docking stations to transfer files to laptops when necessary.

 

slide 2 of 8,zoom in, duplicate or dock a sata ssd/hdd with fast performance - works with both usb-c and usb-a enabled devicesOr buy a $100 Hard Drive docking station and drive duplicator to transfer files to laptops and to easily back up your drives.

 

For robustness, your backup drive should not be used as a hot-swap mobile drive. Either mount them directly in a desktop computer normally used for analysis or duplicate on a weekly basis and then leave them in a safe location.

Panic

Between the three of these solutions, you should be able to deal with acquiring upwards of 10-50 TB a year, but if you are acquiring over 200 TB a year, then it’s time to panic.

100 TB only requires ~$5000 worth of personal drives and backups, but you are hitting the point where drive failures are a part of life. Storing the drives and making sure the backups haven’t failed will be a lot of work. Talk to IT again, and you might want to call up some companies and maybe just stop and think about how your going to analyze all that data anyways.

I hope this has been helpful, and I’m sure many people will disagree with my suggestions here, to summarize:

 

Thanks to Dr. Walter Schwenger, Isaiah Laderman, and Dr. Marc Ridilla for much of the information in this post.

Technical Tuesday: Protein Purification on a Budget

Welcome to the first Technical Tuesday! Where we try to dive a little deeper into the actual costs involved in particular scientific techniques. We hope to show where you can save money, and what you might lose by saving that money. Today’s topic is protein purification by columns thanks to Dr. Marc Ridilla who provided most of the information and all the graphics in this post..

Nowadays purifying proteins with columns on an AKTA allows for a lot of control in flow, buffer mixing, and data collection. Yet AKTA’s are expensive and often that sort of feedback isn’t necessary.

Either way, you might consider making your own columns. A 5 mL HiTrap TALON column goes for over $100 right now, while TALON media comes it at a similar price every 10 mL.

You can buy a C Column for a few hundred dollars to use with the Talon medium, and this gives you more flexibility, you can now create columns of variable volume. If you don’t intend to use an AKTA, you might as well just use PD-10 columns.

Which brings us of course to the real point, in that an AKTA is necessarily the best way to run a column a lot of time. As far as cost goes, the cheapest way to run a column, is you (or gravity).

and the cheapest way to collect fractions, is you.

Of course, if you are collecting fractions manually and pumping manually you will lose a lot of time. We think a happy middle ground consists of a peristaltic pump attached to self-made columns. This gives you a lot of control and you can get precise scientific protein purifications for just $500

The benefit to this approach is you already have AKTA compatible columns if you decide to spring for something like that later. If you buy (or make) a double set of programmable pumps you’ll even be able to run elution gradients.  Though this will cost you a bit more money.

Dr. Peter Foster also notes that by working with syringe pumps directly instead of an AKTA you have no dead volume, which means less time in each step which means less time for those tricky proteins to degrade.