Hmm, well I've seen several errors. Main common ones on the technical side of things are cross contamination, omitting reagents, mixing up which tube is which, insufficient mixing the tube, adding in WAYYYY to much enzyme due to the glycerol content.

Then there are gel loading issues (poking holes in wells, mixing up tube/well order). Loading in ladder as loading dye into all samples (that was a pricey gel!)

Really, the main challenge I see in understanding what is happening and predicting results. I task lab students with sketching out the predicted gel image while the gel is running.

Is this for a larger teaching lab? If so . . . aliquot out everything so no one contaminates the plasmid stock.

Thank you Dr. Amy Klocko for your response. I am generally just collecting information so I do not repeat mistakes from previous undergrad fellows. I am in a lab and we are about to get ready to do plasmid digestion/purification and I just want to be ahead by prepping myself. The last thing I want is to be under prepare and waste reagents and resources .

Uh, well... Mixing up milliliters (ml) and microliters (ul). One of the students has added 1ml of restriction enzyme instead of 1ul. Luckilly that was one of the cheapest enzymes in the box, bought in bulk.

And yes, the digest was successful, I've seen the photo of the gel.

The second mistake typical for students: using tip more than once. Typically lead to cross-contamination of samples or contamination of stocks. Tip is cheaper than the ruined experiment. I make an aliquots of everything for students' training.

Hello Christopher,

Ah, I was thinking you were adding a digest to a teaching lab. Just checking things for yourself will be a much simpler task. Ok, here is my advice.

Get a nice recipe written out, be sure to switch tips (every time you pipet anything, even if it's the same liquid just into a different tube), label your tubes, start with adding the water and buffer as those are cheap, then DNA, then enzymes last. Mix well! Check off the reagents as you use them, and move the tubes (like front of ice tub to middle) each time to add something to help you keep track of which ones you finished that addition.

You'll need an uncut DNA control that you set up just like your digests (buffer, water, DNA) and incubate along with them.

I also will suggest digesting twice what you think you'll need and then run half your digest on your gel (just in case, I've dropped a few gels myself)

Sketch out a picture for what you predict your results will look like then compare to the actual gel.

Good luck and hope it all works out fantastic.

Thank you Dr. Klocko. I appreciate your advice and I will do my best!

not documenting all of the conditions so when you go to empirically troubleshoot you don't know what to adjust. I would say educate them on the importance of details and the important aspects of the digest - salts, temp, time, enzyme amounts, input, etc

Hello Christopher

My most important advice is to be patient and document everything. Even a negative result can be good if you learn from it.

One of the biggest issues I have seen is in the quantity of reagents used. I recommend you write down your protocol and calculations in your lab notebook first to ensure the math is right. Then make a master mix of your rxn +1 adding everything into a single Eppendorf except the sample (enzyme, buffer, water). Then label your rxn tubes. Place your required amount of master mix into the rxn tubes you can use the same tip for this. Then add your sample to be digested. The things to remember are to aliquot slowly to avoid turbulence and get air instead of sample. I know everything comes as a kit nowadays and you follow the protocol but if you want to stand out learn about how the kit works. What is the purpose of the enzyme, buffers, time, temperature, why those amounts, where does it cut and how big should your fragment be? I always have my students start with a plasmid and with restriction sites and follow the sequence to identify the sites they want and think about downstream applications, what will you do with the fragment and remaining vector after digestion?

Not having into account the methylation status of the target, particularly when the target is methylated by overlapping has happened to me at the beginning. Also not putting a positive control of the digestion so you can be sure that the enzyme is working or loading an undigested control in your gel.