Plasmid Extraction using MiniPrep

After making cultures of bacteria with the desired plasmid and allowing the bacteria to grow, it was now time to extract the plasmids. This was done using a kit called MiniPrep. This was the first time I had used this kit, however I have previously used a MidiPrep kit to do a similar process.

Overnight Bacteria cultures in incubator

To carry out the MiniPrep, cultures were centrifuged at a high G force to seperate out the bactceria and the LB broth. Broth was then discarded and bacteria was resuspended in the provided mixtures in the MiniPrep kit. This kit allows for three main steps:

1. Resuspension - First step of the MiniPrep kit, where as mentioned before, allows the resuspension of bacteria into a liquid to allow the next steps to occur.

   
   

2. Lysis - This step allows the bacteria to be broken up to release the plasmids. As the cell is opened up, other cell debris is produced and needs to be removed!

   
   

3. Neutralisation - Neutralises the solution from the lysis step, and allows easier seperation of plasmid.

4. Binding - Binds the plasmids to a column to allow other cell debris to be removed.

5. Wash - Removes extra proteins/salts/other impurities

6. Elution - The final step, where our plasmid is released from the central column and collected into eppendorf tubes.

Mini Prep plasmid extraction!

Again as this kit was straight forward and easy to follow I faced very little difficulties during this procedure. However, unexpectedly on the final step of the kit, some tubes appeared to have more volume than others suggesting that I may have pipetted wrong or faults with the column - which could be either!

Despite this, all plasmids were then taken to the Nanodrop and concentrations of plasmid DNA was recorded. With the concentrations now known, I was able to send my plasmids off for sequencing to ensure that the plasmids have the correct sequence.