We noticed purification using a miniprep kit yields insufficient concentrations of DNA, therefore we adapted our own protocol using the reagents supplied with the GeneJET™ Plasmid Miniprep Kit. We noticed that our optimized purification protocol yields dramatically increased concentrations of DNA.
Please note that while we could have prepared our own reagents we decided it was easier to just use the reagents supplied with the Fermentas miniprep kit.
We used the following reagents from the Fermentas miniprep kit:
- Resuspension buffer
- Lysis buffer
- Neutralization buffer
- Elution buffer
- 70% ethanol
- Check if the RNase A solution has been added to the resuspension solution.
- Keep the resuspension solution cold.
- Check lysis and Neutralization buffer for salt precipitation.
- All centrifugations should be carried out at >12000 g
- Use 1-5 ml E.coli culture in LB media for purification of high copy plasmids.
- Pellet 1-5 ml of culture by centrifuging 5 minutes in 2ml Eppendorf tube. If the volume of the culture exceeds 2 ml, first pellet 2 ml, decant supernatant, apply the rest of the culture to the eppendorf tube and centrifuge again.
- Resuspend the pelleted cells in 250 µl of the resuspension solution. Pipeting up and down till the cells are completely resuspended. (There is no pellet left). Note: Ensure RNase A has been added to the resuspension buffer.
- Add 250 µl of the lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Note: Do not vortex, do not incubate for more than 5 minutes.
- Add 350 µl of the neutralization solution and mix immediately and thoroughly by inverting 4-6 times.
- Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
- Decadent the supernatant in a new 1,5 ml tube.
- Add 650 µl of isopropanol. Invert 4-6 times.
- Centrifuge for 5 min.
- Remove isopropanol by decanting.
- Add 500 µl of 70% ethanol, and remove by decanting.
- Let the pellet dry. You can remove some residual ethanol by pippeting. Note: It is essential all the ethanol is removed.
- Resuspend the pellet in 50 µl water or Elution buffer. Note: This is just a tris-buffer so it will not interfere with the ligation.
- Check the concentration of the plasmids on agarose gel or with a Nanodrop.