User:Kfifer

Lab Notebook

June 14, 2006

Plasmid Miniprep

Samples

• lac operon promoter
  • R0010-1
  • R0010-2
• promoter and GFP
  • E0241-1
  • E0241-2
• GFP
  • E7104-1
  • E7104-2

Qiagen Miniprep Protocol (from kit essentially)

1. Remove growing LB cultures from warm shaker.
2. Set aside 1mL of each sample for later glycerol storage at -80 if the transformation worked.
3. Use an eppendorf tube to spin down the rest of the LB culture (probably in about 3 spins each - keep the pellet each time and remove the supernatant by just pouring it off. The final time, pipet the supernatant away so that there's nothing left but pellet.)
4. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. Pipet up and down. No cell clumps should be visible after resuspension of the pellet.
5. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
6. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
7. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
8. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
9. Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results.
10. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
11. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 60 s.
12. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
13. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
14. Use the nanodrop to determine the concentration of the DNA.