I get many people complaining to me about poor DNA yields from commercial plasmid plasmid prep kits. In this article I will explain the main pitfalls in plasmid isolation and how to avoid them.
Get the culture prep right
You will always get the best results by taking care in preparing the culture. Innoculating from old colonies or allowing the culture to become too saturated will result in poor plasmid replication and retention, which could severely affect the yield. In the past grew my midiprep cultures overnight so they were completely saturated in the morning, but since I switched to the following procedure, my yields have improved dramatically:
- Start with a fresh colony, no more than a few days old.
- Innoculate a 5 mL LB starter culture with the colony in the morning and grow until the OD600 is approximately 1, then store in the fridge overnight.
- If you are using ampicillin as the selection vector, pellet the cells and resuspend in fresh, antibiotic-free medium to remove the secreted beta-lactamase.
- Next morning innoculate a 100 mL LB culture with 1 mL from the starter culture for high copy number plasmids, double the volumes for low copy plasmids.
- Grow the culture until OD600 is around 3, then pellet the cells and proceed directly to the miniprep, or freeze the cells at -2o°C and continue at a later time.
Make the lysis and neutralization thorough
The biggest mistake people make in midipreps is with the mixing in the lysis and neutralization steps. The protocols normally emphasize the importance of being gentle to prevent shearing of the genomic DNA, but I find that people tend to be too gentle. Another common problem is that the lysis allowed to proceed for too long, resulting in permanently denatured, undigestable DNA. Here’s how I approach lysis and neutralization:
- If you are using a low copy number plasmid, use double the recommended volumes of re-suspension, lysis and neutralization buffers. This might also help for high copy plasmids that have given low yields or poor lysis in the past.
- After adding the lysis buffer, instead the 4-6 inversions recommended in the protocols, gently mix by inverting the tube continuously for 3 minutes then immediately add the neutralization buffer.
- Mix the neutralization buffer by gently inverting continuously for 1 minute.
- If the precipitate looks like desiccated coconut, then things are looking good. If it looks gloopy, this is bad. Try to break up the gloop by mixing, this time a little more vigorously (but still quite gently) for a further 1 minute.
- If the glop still does not break up, then the yield is likely to be poor. Next time use double buffer volumes as described above.
Protect the column, and elute twice
Just a couple more quick tips.
- If you are using centrifugation to remove the precipitated debris, filter the supernatant though Whatmann paper before applying it to the column since small amounts of precipitate could easily block the column.
- Perform the elution twice (into separate collection tubes) since a lot of DNA can be left on the column after the first elution. Using elution buffer heated to 50°C can also help to increase elution yield.