Here are some tips for optimization when using the Monarch Plasmid Miniprep kit. We will start with some important things to remember.
1. The first thing that you want to do when using your kit is to add ethanol to the Monarch Plasmid Wash Buffer 2. Follow the instructions on the bottle's label.
2. Also as soon as you receive your kit, please place the neutralization buffer containing RNase at +4°C for long-term storage.
3. Please keep in mind that the amount of plasmid DNA that you can obtain from a culture depends on the strain of E.coli used, the culture conditions including the media used, and the plasmid copy number.
4. Be careful not to use cell culture volumes that are larger than those recommended, as this can limit the efficiency of lysis resulting in lower purity and lower yields.
5. Resuspend your pellet completely. Any bacteria that haven't been resuspended won't lyse efficiently and the amount of DNA that you will obtain will be lower.
6. When lysing the cells invert the tube several times so that the solution becomes viscous and uniformly darker pink.
7. Make sure that you add the elution buffer to the center of the matrix without puncturing it. The elution buffer needs to be in contact with the column matrix for efficient solution.
8. Be sure to incubate new elution buffer in the column for a full minute before spinning.
9. For plasmids that are larger than 10 Kb heating the elution buffer to 50°C prior to adding it to the column can improve yields.
Tips for isolating pure DNA
10. To avoid genomic DNA contamination in your sample, be sure not to vortex the sample after you resuspend the cell pellet. This can cause DNA sharing and consequently genomic DNA contamination.
11. To avoid DNA denaturation during the cell lysis tap do not incubate samples longer than one minute.
12. It is important to avoid RNA contamination. When neutralizing the solution, invert the tube sufficiently so that the solution is uniformly yellow. This allows for a complete RNase treatment of the sample.
13. Make sure that you incubate the sample for a minimum of 2 minutes. This will guarantee complete degradation of the RNA in your sample.
14. When removing the column from the collection tube after the last wash, make sure that the tip of the column does not touch the walls of the collection tube. If you observe that the tip of the column did come into contact with the collection tube then we would recommend an additional 30 second centrifugation step prior to placing the column in the 1.5 ml microcentrifuge tube for elution.You can skip the 2 minute incubation after neutralization.
Tips for saving time
15. If the cell debris pellet is compact enough that it won't be inadvertently transfer to the column you can reduce the spin time after neutralization to 2 minutes.
16. After using wash buffer 1 and your binding your sample to the column you can spend for 30 seconds rather than 1 minute.
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