PCR Hacks

​If you do bench work in a life science lab, you’ve likely delved into molecular biology techniques (PCR, cloning, site-directed mutagenesis). Ask any scientist; they have a love/hate relationship with it. Love it when it works, hate it when it doesn’t.

Most biologists use PCR for two reasons:

1) Cloning: Amplifying an insert piece of DNA to subsequently cut with restriction enzymes and paste into a plasmid.

2) Quick changes (site-directed mutagenesis): Introducing a point mutation, small insert, or deletion into an intact plasmid

As a fifth-year PhD candidate, I’ve done my fair share of cloning and site-directed mutagenesis. Not to toot my own horn, but my success rate is pretty high. Why, you may ask? Here’s why:

1. Primer Design Hacks

• For site-directed mutagenesis, use non-overlapping primers: I’ve had consistent success with non-overlapping primers. Place your mutation at the 5’ end of the primers (don’t overlap the mutation either!) Only two primers are required per mutation. This method requires subsequent phosphorylation and ligation steps after the PCR. See below for an example of designing site-directed mutagenesis primers where two amino acids are undergoing mutation.

• Walk away and then double-check: Once you have designed your primers, don’t order them right away! Save your primers, then come back an hour or even a day later. You’d be surprised what mistakes you catch with fresh eyes!
• Design more than one: Primers are only $5 each, so if you’re on the fence about doing a longer or a shorter primer, or if you’re not sure if you should design the higher or lower temp, order both! Run the PCRs side by side and save yourself time. 

​2. PCR Hacks

• Use fresh water: Water can accumulate contamination over time, so to be careful, pour yourself some fresh ultra-pure water each time you do a PCR.
• Buy the master mix: Buying pre-assembled master mix is a major time-saver! No more assembling the mix, running quality control checks, or throwing away bad mixes. Most enzyme companies sell premixed 2X master mix at a comparable price to buying the master mix reagents separately.
• Always run a gradient: Annealing temperatures of primers are easily estimated by online tools or the manufacturer. But be aware that the estimated temperatures are not always accurate. Run three reactions per primer set. One temperature at the estimated annealing temperature, one reaction 5° higher, and one reaction 5° lower. Go forth with the phosphorylation, ligation, and plasmid prep steps for all PCR reactions that work to increase the likelihood of getting the desired product. If no reactions work, try one more time at 10° lower before redesigning your primers.

3. Cloning hacks

• Use more DNA: Higher concentrations of DNA can increase the likelihood of proper digestion and ligation. Start your cloning process by plasmid prepping fresh vector and eluting in ½ the recommended volume so that you have a concentrated sample. When amplifying your insert, amplify for 33-35 cycles. And lastly, when digesting the insert and vector, use 2X the amount of recommended DNA.
• Didn’t work the first time? Do it again. Human error in cloning is common. Make a checklist and be super focused to rule out human error while cloning.

4. Plasmid prep Hacks

• More volume = more DNA: Use 2x the recommended broth volume for your overnight culture. Still have small DNA yields? Use even more volume.
• Overnight more than one colony:  After transforming your ligated vector, you’ll end up with a plate of a bunch of colonies. Some of these colonies will have your desired product, and a few will not. For quick changes, set up 2x overnight colonies per plate (in two separate cultures), and for cloning, overnight 4x colonies per plate.

So there you have it! My tips for increasing your PCR success rate. Give them a try!!

Author

Kerry McPherson, @KerrySilvaMcph 
PhD Candidate studying biomedical sciences. Researches proteins implicated in cancer chemoresistance. Bolded Science creator and editor. Also co-founded a STEM education outreach program at her University.