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Team:Amsterdam/Notebook/Protocols/Miniprepping
From 2011.igem.org
Miniprep protocol
We stopped using the [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf GeneJET™ Plasmid Miniprep Kit protocol]. We noticed that the optimized purification protocol gave much better results. With this protocol the yield increased dramatically.Reagents
We used the following reagents from the Fermentas miniprep kit:
- Resuspension buffer
- Lysis buffer
- Neutralization buffer
- Elution buffer
Other reagents:
- Isopropanol
- 70% ethanol
Notes:
- 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.
Protocol
- 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 tub 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.
