| Large Clone Isolation Protocol |
| Version Number: | 1 |
| Start Production Date: | X |
| Author: | Jason Olivas |
| Edited by: | X |
| Reviewed by: | Amy Geotina |
| Summary |
| Materials & Reagents |
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| 50-ml pipettes | Falcon | 357550 |
| 25-ml pipettes | Falcon | 7525 |
| 10-ml pipettes | Falcon | 357551 |
| 5-ml pipettes | Falcon | 357543 |
| 1-ml pipettes | Fisherbrand | 13-678-11b |
| Stock Solutions | ||
| DDD Water | JGI | N/A |
| 70%Ethanol | JGI | See Below |
| Tris-EDTA | JGI | See Below |
| Reagents | ||
| Qiagen Maxi-prep Kit | Qiagen | 12165 |
| SDS (Lauryl Sulfate) | Sigma | L-4522 |
| 1N NaOH | Fisher | SS266-1 |
| LB Broth | Teknova | 0181-iL |
| 100% Isopropanol | VWR | PX-1835-14 |
| Equipment | ||
| 250 ml Centrifuge bottles | Nalgene | 3141 |
| 50 ml Centrifuge tubes | Nalgene | 3139 |
| Sorvall/Beckmen Centrifuges | Sorvall/Beckmen | N/A |
| Procedure |
Inoculation:
1. In a 500ml flask, pour 250mls of LB Broth.
2. Add appropriate antibiotic: Chloramphenicol (15ug/ml) or Kanamyicin (25ug/ml).
3. Inoculate with 10-20 uls of glycerol stock for one unique clone.
4. Grow for 18-20 hours in the Innova 4900 upright shaker at 37 C at 220 rpms.
Isolation-modified protocol of the Qiagen Plasmid Maxi-Prep protocol
1. Set a timer for 5 minutes and one for 15 minutes.
2. Pour culture into 250 ml centrifuge bottle. Pellet cells at 4,000 rpms for 15 minutes at 4 C.
3. Pour off supernatant into waste beaker. Add 50% bleach solution to supernatant to kill leftover bacterial cells.
4. Resuspend cell pellet in 12 mls of P1 (Resuspension) buffer. Add RNAse to P1 buffer so that the final concentration of the P1 buffer is 100 ug/m1. Make sure no cell clumps are left over.
5. Add 12 mls of P2 (lysis) buffer to the resuspended cells. As soon as you add the P2 buffer to the very first bottle, start a timer for 5 minutes. DO NOT shake or mix the centrifuge bottle since this may lead to a higher E.coli percentage.
6. At the end of 5 minutes, add 12 mls of chilled P3 (Neutralization) buffer to the lysed material. As soon as you add the P3 buffer to the very first bottle, start the 15 minute timer. Should lyse cells no longer than 15 minutes.
7. Pour the contents of the 250 ml centrifuge bottles into 50 ml centrifuge tubes being careful to not shake and mix as little as possible in the transferring. Put on ice until the 15 minute neutralization step is completed. Steps 6 and 7 should all occur within the 15 minute neutralization period.
8. At the end of the 15 minute neutralization period, centrifuge the samples at 15,000 rpms for 30 minutes at 4 C.
9. While the 30 minute spin is proceeding, set up the appropriate amount of Qiagen columns.
10. After the 30-minute spin is completed, transfer the supernatant to a clean 50-ml centrifuge tube and spin at 15,000 rpms for 15 minutes at 4 C. While spinning, equilibrate the Qiagen columns with 10 mls of QBT (Equilibration) buffer. Allow the solution to empty out of the columns by gravity flow.
11. After the 15-minute spin, pour the supernatant onto the columns and allow it to empty by gravity flow.
12. Wash the columns with two (2)-30ml aliquots of QC (Wash) buffer. Allow emptying by gravity flow.
13. Elute the DNA from the columns into a clean 50-ml centrifuge tube with heated (65 C) QF (Elution) buffer. Add 8mls of the heated buffer then reheat it while buffer is eluting out the DNA. Add another 7mls of reheated buffer to finish off the elution.
14. Precipitate the DNA with 10.5mls of 100% room temperature isopropanol. Mix and centrifuge at 12,000 rpms for at least 30 minutes at 4 C.
15. Carefully pour off the supernatant as not to disturb the precipitated DNA pellet.
16. Wash the DNA pellet with 70% ethanol by spinning the pellet with the ethanol at 12,000 rpms for 15 minutes at 4 C.
17. Carefully pour off the ethanol without disturbing the DNA pellet.
18. Air dry the pellet in a vent hood for 30 minutes or more until the majority of the ethanol has evaporated. Angle the tubes so that the ethanol is as far away as possible from the DNA pellet as possible.
19. Resuspend in 1ml (1000 uls) of filtered TE overnight in a deli refrigerator at 4 C. Angle the tubes so that the TE completely submerges the DNA pellet and ensures that the whole pellet resuspends back into solution. Anchor your tube rack so that the rack does not tip over at all when resuspending overnight.
| Reagent/Stock Preparation |
70% Ethanol-Make 500 mls. Use 350 mls of 100% ethanol and 150 mls of DDD water.
Lysis Buffer-Make 250 mls. Use 175 mls of DDD water, 50 mls of 1N NaOH, and 25 mls of SDS (Lauryl Sulfate). Make fresh before starting the large clone isolation procedure.
P1 Buffer-add RNAse (100mg/ml) to the P1 buffer solution before starting the large clone isolation procedure. RNAse once added to the P1 buffer, needs to be refrigerated at 4 C.
P3 Buffer-make sure P3 buffer is ice cold before starting the large clone isolation procedure.
Chloramphenicol Preparation-add 0.51 grams of chloramphenicol to 15 mls of 100% ethanol for a 34 mg/ml chloramphenicol solution.
Kanamyicin Preparation-add 0.6 grams of Kanamyicin to 30 mls of DDD water to make a 20 mg/ml kanamyicin solution.
TE- mix 1ml of 1M Tris pH 8.0, 200 uls of 0.5M EDTA pH 8.0, and 998.8 mls of DDD water.