Plasmid endo-free maxiprep

Reagents

The DNA prepared can be used for ES cell transfection and transgenic mouse injection, etc.

1. Day 1, seed single colony of bacteria into 3 ml LB and culture for 8 hrs at 37C, RPM 220.

2. Transfer the bacteria into 100 ml LB (1:1000) and culture at 37C for overnight (12-16 hrs).

3. Day 2, pellet bacteria by centrifuging at 6000 RPM for 10 min at RT, discard supernatant.

4. Invert the tube on paper towel and drain all remaining LB media from the pellet.

5. Add 10 ml buffer P1 (from 4C) and vigorously mix the pellet, make sure no visuable clamps left.

6. Add 10 ml buffer P2 and quickly invert the tube gently for 4-6 times, leave at bench for 5 min.

7. Add 10 ml buffer P3 (from 4C) and quickly invert the tube gently for 4-6 times.

8. Transfer all solution into a cartridge and leave it on bench for 10 min. All cloudy precipitates should float to the top.

9. Remover the stopper and pass the solution through the cartridge using the plumger. You can get ~25 ml solution.

10. Add 2.5 ml ER buffer into the flowthrough and vigorously mix it, keep on ice for 30 min. The solution becomes turbid and it will be clear soon.

11. In ther waiting time, prepare column and add 10 ml QBT buffer into each to equilibrate the column.

12. Add the DNA containing solution from 4C to the column by dumping.

13. After draining, add 30 ml buffer QC to wash the column. Repeat one more time. You do not have to measure 30 ml QC but just dump to the column to the top which is 30 ml.

14. After washing, put the column into a new clean tube add 15 ml QN buffer to elute all DNA from the column.

15. Add 10.5 ml 2-propanol and mix it immediatedly by inverting the tube for 10 times.

16. Spin down at 4C for 1 hr at 4000 RPM.

17. Carefully remove supernatant and keep the DNA pellet in the bottom of the tube.

18. Add 5 ml 70% ethanol into the tube and shake it for a while.

19. Spin down at 4000 RPM for 1 hr at 4C.

20. Remove the supernatant carefully.

21. Air dry for 5-10 min until the pellet becomes transparent.

22. Add 200-500 ul TE to dissolve the pellet.

23. Transfer the DNA into a clean Eppendorf tube and spin down at 14K RPM for 1 min.

24. Transfer the supernatant into another clean Eppendorf tube and that is the DNA.


Comments

Add a comment
Posted by ebiomethods Tue, 23 Dec 2008 10:21:50
Subject: Comments are welcome
Thank you.
Site News
New feature:
You can add comments on protocols now. Just go to any individual method page and click on the "Add a comment" link.

Highlight of the month
Autophagy hits heart

Autophagy is acutely upregulated to provide necessary nutrients during starvation for survival. Under normal condition, constitutive autophagy is critical to perform house-keeping functions to eliminate damaged organelles and dysfunctional long-lived proteins.

Ageing is associated with accumulation of dysfunctional proteins in cells. Autophagic activity is decreasing along with ageing. Studies in worms and flies show that reconstitution of autophagy can increase life span. A recent report by Taneike and colleagues explored the role of autophagy in age-related cardiomyopathy.

In heart, autophagic activity is decreasing when ageing. Taneike et al eliminated autophagy in heart by knocking out an essential gene, ATG5, specifically in heart. The researchers found deteriorated cardiac function at 10 months of age in the deficient animals compared to wild type controls. The dysfunction of autophagy causes accumulation of dysfunctional mitochondrial, reduced mitochondrial efficiency and significant oxidative damage in cardiomyocytes. More interestingly, evidence of mitochondrial damage is obvious as early as 3 months old, before cardiac remodeling and dysfunction manifest. This study points to a possibility that autophagy is critical in maintaining cardiomyocyte homeostasis and autophagy may be a target for future therapeutic design for heart failure.... Read more highlights.