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October 2015 Archive

Posted by Karen on October 14th, 2015  ⟩  0 comments

Choosing the right biological buffer for your experiment can be tricky. The buffer choosing process is like playing “Guess Who.” Like the game, you begin with a very broad list of buffers and slowly ask questions and research answers to narrow down your selection.

                                                                                                            

By now you know all about Good’s Buffers and are looking for more guidance to help with this little game. Well, in this article, I’ve cut to the chase – scrapping the talks about Good’s Buffers and getting right into the other things you should consider. For more details about choosing the right buffer, troubleshooting and other information about biological buffers, check out our Buffer User Guide. It doesn’t cost a thing and it is loaded with all the information you need.



Step 1: Determining pH for your experiment

In many experiments, you’ll need to change conditions in order to keep your proteins happy in solution. Let’s say, for example, you’re doing an experiment at pH 7.4, which mimics biological conditions, but your protein isn’t stable at this pH, you’ll need to optimize the situation. A good thing to keep in mind is that your target pH is usually going to be about one pH unit away (sometimes slightly farther) from the protein isoelectric point (pI). The pI is the pH where a given molecule or protein carries no net charge. When a protein is at its pI, it isn’t quite as soluble. Calculating pI on your own can be a bit of a challenge. But don’t worry; we live in the age of the Internet, and there are plenty of online calculators out there such as the one from ExPASy.



Step2: Considering Unwanted Buffer Interactions

Another important step in deciding which buffer to use is determining if there would be any negative buffer interactions. For example, if you’re working with an enzyme, your organic buffer might influence enzymatic function for better or worse.




  

Step 3: Asking the Right Questions

Just like the game, you have to ask the right questions to be certain you’re getting the right buffer. While we have several listed in the Buffer User Guide, I’ve highlighted a few great questions here:

1.Are there any metals in solution which a buffer might complex with?

2.Is your target protein an enzyme, and what class does it belong?

3.What are the ionic strength requirements for ligand binding, protein stability or electrophoresis?

4.What literature exists that tells me about interactions of my proposed buffer and the system I am using?

All of these questions, plus the others listed in our guide are going to further help you find the most appropriate buffer for your experiment.



Step 4: Considering Downstream Experiments

One stumbling block that often happens for researchers is that they’ll start with a buffer and find out that it won’t work for downstream applications. For instance, Tris has been known to negatively interfere with the Bradford assay. Other biological buffers can negatively impact mass spec. Unfortunately, after steps 1 and 2, you have probably narrowed down your buffer only to find out it might not work so well down the road. It’s like a nightmarish version of “Guess Who.” In this case, you’ll have to do some research in finding appropriate alternatives; however, now that you’ve narrowed down the characteristics you need, doing that research will be a little easier.



Step 5: Sourcing the Right Buffers

Great, now you know exactly what buffer you’re going to use. It’s time to source your buffers. For most researchers, bottom line and purity are basically the most important criteria when it comes to where to buy buffers. Sure you can go to the top name guys and know you’re going to get the right product, but you also know it won’t be for the right price, even if you’re making your own buffers. Instead, GoldBio has a growing catalog of high-quality powder buffers and the price is exceptional.

Not only does GoldBio offer great products, but the products are backed up with excellent support and content to help you with your experiment. It’s further evidence of a company you can trust.

Be sure to check out our Printable Buffers Selection Guides – Arranged by pH or by Family!

how to choose the right buffer - Buffer selection guide by pH

Buffer Selection Guide Arranged by pH. Print, and
hang at your bench today!
                      
How to choose the right buffer for my experiment - Buffer selection guide by Family

Buffer Selection Guide Arranged by Family. Print and
hang at your bench today!


    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science. 



Category Code: 88253 79107

Posted by Karen on October 29th, 2015  ⟩  0 comments

Press down. Cross fingers. Squint eyes in anticipation – annnd SNAP! Electroporation failed again. Electroporation is one of those techniques that can feel almost confrontational. You’ve been careful about salinity. You’ve avoided the bubbles, but that snap just aims to taunt you until you develop a complex.

Ok, so what do we do about this? First of all, if this is happening to you and you’re constantly frustrated, you’re not alone! As is common with my articles, I’ll take you through a few solutions that might possibly help you get the upper hand in the dreaded electroporation.


1.Desalting DNA:

As I mentioned before, you’re probably very careful about the salinity but a paper by Christina Schlaak and her team explains that your desalting method after ligation really matters. According to the paper, given the electric field and size of the cuvette, it doesn’t take much ligation mixture to cause your electroporation to fail. While a lot of researchers will point to drop dialysis or ethanol precipitation as the choice for desalting, Schlaak’s research found that microcolumn purification is in many cases the more effective approach.



2.Ice Your Cuvettes Right Before:

It’s pretty self-explanatory, but remember to keep your cuvettes cold.



3.Store Cuvettes in the Freezer:

Some researchers swear by having cuvettes as cold as possible. This means storing cuvettes in the freezer until they’re ready for use, keeping them on ice and even icing for a few minutes before electroporation.



4.It Could Be a Matter of Concentration:

So maybe you think that your DNA is too concentrated. And that’s possible, but another possible situation is that your cells are too concentrated. You might want to consider diluting your cells and trying again.



5.Glycerol Impurities:

This doesn’t happen often, but it’s possible that impurities in glycerol raise the conductivity and cause your electroporation to arc.



6.Understanding Bubbles

Of course you do the bench tap to minimize those bubbles, but it’s a good idea to have in the back of your mind what the reason is. Think of your electroporation like a strike of lightning. The liquid in your cuvette provides less resistance and allows for a more direct shock. Air bubbles reduce that direct impact, causing an uncontrolled shock to occur. So tap out those bubbles and be sure not to have any splatters.



7.Check Your Voltage Against Your Cuvette Size:

The size of your cuvette is going to be a factor when optimizing voltage and field strength. For instance, let's say you're used to using a 2mm (.2cm) gap cuvette  with a voltage of 900 and a field strength of 4.5 kV/cm but for some reason, for this experiment, all you can find is a 1mm cuvette. You will have to change your settings to maintain the same field strength. In this case, you would want half the voltage or 450V. 



8.Check Your Electroporator Against the Protocol:

The best analogy here is the classic case of microwaving and then burning your popcorn. The bag’s instructions tell you, for example, to microwave it for 2:30, but it always comes out with more kernels and less popped corn. If you have a popcorn button, you’re supposed to use that instead, and even that doesn’t work. So you have to sacrifice a few bags to figure out the best settings. Likewise, the settings in your protocol might be for a specific electroporator, which differs from yours. This means you might want to try tweaking your method to get it just right.




9.Check Your Competent Cell Preparation:

If you’re making your competent cells and time after time, your electroporation arcs, then it might be the result of your competent cell preparation.



10.Don’t Touch the Aluminum Electrodes:

Be mindful about the location of the electrodes. Touching them can increase the sample temperature which puts you at risk for the dreaded SNAP.



11.Check Your Cuvettes for Cracks or Aging:

Old cuvettes could be your problem. Check them for cracks in the plastic.



12.Snap Doesn’t Always Mean the End:

This is more of a last resort to consider, especially if you’ve burned through everything. Even if your electroporation arced, it’s possible that you might still have a transformed clone.



With 12 options here, you might wonder, “where should I start first?” Let’s begin by categorizing these steps into: Preparation Best Practices, Execution Best Practices, Variables/Tweaks.

Preparation Best Practices: These are things you might not be able to fix for a sample in process, but you can adopt for the next go around.

Execution Best Practices: These are tips that you want to make sure you’re following during the electroporation process; however, in some cases it might be too late for a sample in process.

Variables/Tweaks: These are the tips you might want to focus on for something you have currently running.

Electroporation Troubleshooting Tip Chart



There you have it. Now you know what to try and when to try it. Now go and conquer your next electroporation!



Schlaak, C., Hoffmann, P., May, K., & Weimann, A. (2005). Desalting minimal amounts of DNA for electroporation in E. coli: A comparison of different physical methods. Biotechnology Letters, 27(14), 1003-1005. doi:10.1007/s10529-005-7867-z




    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
college, and the revelation that the adults in my past were right all
along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
and loving everything science. 



Category Code: 88253 79103 79108 79107