What do we refer to when we talk about minipreps?

The most common minipreps are from DNA. in our case we took out a plasmid of a
bacteria, while taking in count the contaminating proteins and genomic DNA we
want to avoid. This plasmid we obtain by the Minipreps we can use it for restriction
analysis, sequencing and cloning.

This can be use it for embryonic inject but before we need to do some extra
procedures of cleanup.

Are there different methods to execute them?

There are different protocols for elaborate a mini-prep, all of them have the same
purpose: purify the DNA of a plasmid, but the steps can be realized by different
methods:
- Cell lysis: it can be mechanical, chemical or by temperature changes.
- Remove of big particles it can be by filtration or centrifugation.
- Purification it can be by fractional precipitation, absorption or by chromatographic
methods.
- Preservation it can be by with a solution or freeze-drying.

Steps when you do a miniprep

1. Collect a bacterial colony in a sterile tube.
2. Incubate at 37 ° C overnight.
3. We will proceed to recover the DNA of the bacteria, putting medium in an
Eppendorf tube for bacteria grow, then we centrifugate it, and the bacteria it
should be on the bottom of the tube.
4. We will centrifugate it again for 1 minute and remove the supernatant, throwing it
in the waste container, the bacteria should continue on the bottom of the
Eppendorf tube.
5. We will apply a buffer and resuspend, without finding any pellet.
6. We will apply another buffer and leaving it at room temperature for about few
minutes and we will move it carefullying to do not make bubbles.
7. We will add a third buffer and put it to spin for a few more minutes.
8. While is spinning we’ll prepare a new tube with a column.
9. Once the centrifuge is finished, transfer the supernatant to the column.
10. We will return a centrifuge for 1 minute.
11. Remove the supernatant, add a new buffer solution.
12. Centrifuge for 1 minute again, remove the supernatant and add another buffer
solution.
13. Repeat the process to follow the membrane very well.
14. We will prepare a new eppendorf tube and pass the column from the previous
tube to the new tube, to recover the plasmid.
15. Add buffer and incubate for one minute.
16. We will spin for another minute, we will remove the column, because at the
bottom of the eppendorf we find the plasmid