This is a basic tutorial for making solutions in a lab at a specific concentration. We’ll look at two different situations: a solid being dissolved into a liquid, and a liquid being added to a liquid. 

Before you start: 

• Determine the volume of solution you need. If you can store the solution for future use, make a large batch or a stock solution so that life is easier. 
• Find everything you need before you start work. There’s no point in making your solution if you don’t have the solute/solvent necessary.
• Give yourself plenty of time. If your experiment is about to finish and you just realized you don’t have a solution, it could ruin the experiment, especially if it’s time-sensitive. 

Now let’s get started!

Solutions involving Solids

If you need to make a solution involving a solid (most likely powdered), you’ll need a scale to measure out how much you need. You have two options: you could use a weigh boat or the container that your solution will be in to mass your solid. Either way, don’t forget to tare before massing!

The golden rule of solution making: A 1% solution is 1 g material/100 mL solution.

Our scenario: I’ve just finished running a Western and need 10 mL of 5% blocking milk, which is powdered milk in PBST. For our purposes, the PBST is already made.

1. We have the volume. Gather the beaker for making the solution, the powdered milk, and the PBST. Tare the beaker on top of the balance.
2. Now to calculate. We want a 5% solution, which would be 5 g material/100 mL. Since we want 10 mL, we simply divide the mass (5 g) and the volume (100 mL) by 10, leaving us with .5 g of milk in 10 mL of PBST.
3. Mass the powdered milk in your beaker. After measuring out .5 g, measure out 10 mL PBST in a graduated cylinder, add to powdered milk, and swirl gently until all powdered milk is dissolved. The blocking milk is now ready for use!

Solutions Involving Liquids

As far as calculations go, this is MUCH easier to do than make a solution involving solids. However, you DO have to take the loss of volume into account when making concentrations. We’ll see that in the scenario below.

Our scenario: I need to run a DNA gel, but there’s no more TAE buffer! Luckily, the lab manager has TAE at a 50x concentration. However, I need to bring this down to 1x. 

1. Since TAE can be stored at room temperature, it’s better to make a large volume for future use (unless otherwise directed). Let’s make one liter. You need to gather two graduated cylinders - one for the 50x TAE, and one for distilled water (which we’ll use to dilute the TAE). 
2. There are a variety of ways to calculate, but I like to use proportions. We need to dilute 1:50, and we have 1 L (for our purposes, we’ll make it 1000 mL).
   • 1 TAE/50 water = x mL TAE/1000 mL water
   • If you cross-multiply (not the official way to say it, I know), you’ll get this equation: 1000 = 50x
   • Solving for x, we get 20, which is how much TAE we need.
3. Start off with 20 mL of 50X TAE in your beaker/bottle/container, and bring up the volume with the distilled water until you reach 1 L. Shake the bottle to mix (DO NOT DO THIS WITH VOLATILE LIQUIDS!!) and you now have 1x TAE!

Tips: 

• If you use a weigh boat and want to get all of the powdered solvent into the bottle, first measure the volume you need and pour ALMOST all of it into the bottle/beaker/tube, whatever you’re using. Add the rest of the volume (a small amount, remember!) to the weigh boat and gently swirl/tap until the solid is dissolved. Pour the rest into your container and you’re pretty much set!
• Some solids (such as BSA, or bovine serum albumin) don’t go into solution very easily. For that, you may need a stir plate and stir bar. Allow the stir bar to align BEFORE turning the plate on or else you may splash solution! Also, most stir plates have heating capabilities if necessary to put the solid into solution. 
• Be aware of what your solution needs to be functional. You may have to adjust the pH, keep the solution cold, or warm it up before use. 

I hope this tutorial was helpful! Questions and comments are always welcome via ask box!

I’d like to apologize for not getting this blog off to a more lively start - I’ve been trying to get ready for my first year of college. 

Next post will be a bit different - instead of a lab protocol, I’ll be making a walk-through on how to make solutions at a specific concentration when the solute is in a solid or liquid form. Basic, but essential to lab work. It’ll have a few good tricks as far as quickly calculating concentration. Hope someone finds it useful! 

The following protocol is for making and running an agarose gel for DNA. Very easy to do and one of my favorite procedures besides immunofluorescence (which I may post later). 

Materials:

• Powdered agarose
• TE buffer (TAE or TBE)
• Flask
• Microwave
• Casting tray (varies with each lab)
• Gel comb with appropriate number of teeth
• DNA samples 
• Loading dye
• Electrophoresis chamber and power supply
• Ethidium bromide 

Directions:

1. To prepare the gel, add the appropriate amount of TE buffer to powdered agarose in a flask with plenty of empty space. To make a 1% agarose gel (standard concentration), add 100 mL TE to 1 mg agarose. Higher agarose concentrations (up to 2%) are typically used for small DNA fragments, while lower concentrations (.7%) are for larger DNA fragments. 
2. Heat liquid agarose (agarose in TE) in the microwave until solution starts to boil. WHILE WEARING GLOVES, remove flask from microwave and swirl gently. Look for chunks of agarose and/or remaining powder. 
3. Reheat in the microwave until solution starts to boil again. At this point, swirl again. The solution should be devoid of chunks or any powdered agarose. If they are present, heat again until the solution appears clear.
4. Let flask cool until it can be handled by (gloved) hands. In the meantime, set up casting tray, making sure that the tray is LEVEL. If needed, use a leveling bubble to check. Insert gel comb in the notches closest to the edge of the casting tray. 
5. Once the flask has cooled, add ethidium bromide in a 1 uL: 25 mL concentration. Too much ethidium bromide may cause the entire gel to fluoresce and be difficult to image, while not enough ethidium bromide wouldn’t allow the DNA to bind properly and fluoresce. 
6. Pour agarose into casting tray SLOWLY as to prevent bubbles. Pour gel until it reaches halfway up the comb. Let gel solidify completely. The gel is solidified when it appears a milky white. 
7. Prepare DNA samples. Depending on the purpose for electrophoresis, you may need to change sample volumes in order to have a standard mass of DNA. 
8. Add loading dye to each sample. Loading dye is primarily used to visualize electrophoresis and allow DNA to stay in the well, so how much you add is variable. 1 uL is a good standard to go by. 
9. Once gel has solidified, place gel in electrophoresis chamber and add TE buffer over it. Pour TE buffer until it covers the gel and remove gel comb. If the wells are sticking out over the surface of the buffer, add more buffer until they are completely covered. 
10. Carefully add samples to wells, making sure to save one well for ladder. Try to avoid sample leaking out of the wells, as they could leak into other wells and affect results. 
11. Cover electrophoresis chamber and plug in power supply. Turn power supply and set voltage (I use 80-125 volts, depending on expected fragment size and time desired). Start gel running. Check that gel is indeed running by looking at current function on the power supply or looking into the chamber to see if there are bubbles at each end. 
12. Check to see that loading dye separates into two different bands (colors may vary depending on type of loading dye used). 
13. Let gel run depending on expected fragment size. If fragments are small (100-200 bp), run only halfway down the gel. If fragments are larger, let the dye run until ~2 cm from the opposite edge of the gel. 
14. Turn off power supply and carefully remove gel (which may slide). Visualize gel with a BioRad imager or transilluminator. Be careful when using transilluminator - UV irradiation may cause sunburn and/or DNA damage to skin. Always view gel with protective glass between you and the gel. If you need to touch the gel, make sure hands and arms are covered and ALWAYS protect eyes from UV exposure. 

Hope this is helpful to anyone who needs it! As usual, ask questions about the protocol or leave comments! 

Below is a protocol for isolating DNA from cells grown in culture (eukaryotic). This protocol is particularly good for those of us who can’t afford QiaGen kits. Yields are slightly lower than with the kit, and purity is not quite the same. Good for gels, most likely not-so-good for anything sensitive like direct sequencing. 

Materials: 

• Cultured cells (preferably grown in petri dish, may have to tweak volumes for flask and/or plate-grown cells)
• .5% Triton X-100 in TE buffer
• Proteinase K and Ribonuclease A (1 ug/uL each)
• 3 M sodium acetate
• Ethanol
• TE buffer

Procedure:

1. Depending on cell type and treatment before DNA isolation, DNA may need to be isolated from media or cells attached to the dish. For cells in media, transfer all media into an Eppendorf tube, spin down in a microcentrifuge for approximately ten minutes at 14,000 rpm, and remove supernatant. For cells attached to the dish, aspirate off all media and proceed.
2. Add 50 uL Triton X-100 in TE. For cells previously in media, simply resuspend pellet in the 50 uL. For attached cells, add 50 uL to a 35 mm petri dish, scrape off cells with a cell scraper, and transfer contents of the dish (with cells) into an Eppendorf tube.
3. Add 1 ul each of proteinase K and ribonuclease A (1 ug/ul). Incubate at 55-60 degrees Celsius for at least one hour.
4. Add 40 uL COLD 3 M sodium acetate and 100 uL COLD ethanol to the sample. Incubate on ice thirty to forty minutes. 
5. Centrifuge the sample at 4 degrees Celsius at 14,000 rpm for ten minutes.
6. Remove supernatant and resuspend pellet in 50 uL TE buffer. The DNA is now purified and ready to be analyzed/stored/quantified. 

My mentor and I came up with this protocol in the course of doing my project for Honors Science Research this year. Ratios were rarely, if ever, below 1.000. Yields were variable due to cell treatment with UVB and PFTa. 

Questions about the protocol? Considerations I need to put in? Please let me know :)  

Please submit anything of scientific interest if you have anything!