TITLE: Qiagen QIAprep Spin Miniprep for cosmids from metagenomic library
AUTHORS: Bonnie Evans

[DESCRIPTION]
Taken from QIAprep Miniprep Handbook (see attachment). Isolating cosmids from metagenomic clones for sequencing or transformations.

[BEFORE_START]
Add the provided RNase A solution to Buffer P1 before use. Use 1 vial RNase A (centrifuge briefly before use) per bottle Buffer P1 for a final concentration of 100 μg/ml. Mix and store at 2–8°C. 
Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume). 
Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37°C. Do not shake Buffer P2 vigorously. 
Close the bottle containing Buffer P2 immediately after use to avoid acidification of Buffer P2 from CO2 in the air. 
Optional: Add the provided LyseBlue reagent to Buffer P1 and mix before use. Use 1 vial LyseBlue reagent per bottle Buffer P1 for a final dilution of 1:1000 (e.g., 10 μl LyseBlue into 10 ml Buffer P1). LyseBlue provides visual identification of optimum buffer mixing, thereby preventing the common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris.

[GUIDELINES]
All centrifugation steps are carried out at 14000 rpm (~16873  x g)) in an Eppendorf 5418 table-top microcentrifuge. Volumes of Buffers P1, P2, and N3 have been doubled as we are using a 10 ml culture volume.


[STEPS]
SECTION: Grow bacterial culture overnight
1. Make up 15 mg/mL tetracycline hydrochloride in water.
SECTION: Grow bacterial culture overnight
2. Dilute 1/1000 in LB broth (final conc. 15 ug/mL).
SECTION: Grow bacterial culture overnight
3. Aliquot 10 mL  tetracycline LB into 50 mL Falcon tubes.

SECTION: Grow bacterial culture overnight
4. Using a sterile inoculation loop, scrape the top of the glycerol stock and inoculate in tetracycline LB.
SECTION: Grow bacterial culture overnight
5. Grow for up to 12-16 hours at 37°C and shaking at 180-200 rpm.

 


SECTION: Harvest cells
6. Centrifuge at 4500 rpm (4347 x g) in the Eppendorf 5920 R centrifuge.
SECTION: Isolate cosmid
8. Re-suspend pelleted bacterial cells in 500 µL  Buffer P1 and transfer to a 2 mL micro-centrifuge tube.

 

SECTION: Isolate cosmid
9. Add 500 µL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not allow the lysis reaction to proceed for more than 5 min. 
 

 

SECTION: Isolate cosmid
10. Add 700 µLBuffer N3 and mix immediately and thoroughly by inverting the tube up to 10 times. 

 

SECTION: Isolate cosmid
11. Centrifuge for 10 min.
 

SECTION: Isolate cosmid
12. Apply 800 µL of the supernatant from step 10 to the QIAprep 2.0 Spin Column by pipetting.
SECTION: Isolate cosmid
13. Centrifuge for 30-60 s. Discard the flow through.
SECTION: Isolate cosmid
14. Repeat steps 11 and 12 with the remaining supernatant, carefully avoiding the pellet.
SECTION: Isolate cosmid
15. Wash the QIAprep 2.0 Spin Column by adding 500 µL  Buffer PB and centrifuging for 30–60 s. Discard the flow-through. 
 

SECTION: Isolate cosmid
16. Wash the QIAprep 2.0 Spin Column by adding 750 µL  Buffer PE and centrifuging for 30–60 s. 
SECTION: Isolate cosmid
17. Discard the flow through
SECTION: Isolate cosmid
20. Heat Buffer EB or water at 70°C in a block heater

 

SECTION: Isolate cosmid
19. Place the QIAprep 2.0 Spin Column in a sterile 1.5 ml microcentrifuge tube. 
SECTION: Isolate cosmid
21. To elute DNA, add 50 uL pre-heated buffer EB or water to the centre of each QIAprep 2.0 Spin Column 

 

SECTION: Isolate cosmid
18. Centrifuge at full speed for an additional 1 min to remove residual wash buffer. 

 

SECTION: Isolate cosmid
22. Let stand for 60 s
SECTION: Isolate cosmid
23. Centrifuge for 60 s
SECTION: Harvest cells
7. Pour off supernatant.
SECTION: Isolate cosmid
24. Measure the concentration of dsDNA in 2 µL of sample using the NanoDrop spectrophotometer