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| META TOPICPARENT |
name="ProtocolList" |
Lambda Red Protocol
Lambda Red Plasmids
- pKD46 (ampR)
- pKD78 (camR)
Making Competent Cells expressing Lambda Red
Day 1 |
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- Grow starter culture (of strain of interest with pKD46/pKD78) in 10mL LB-Amp overnight at 30°C
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- Grow starter culture (of strain of interest with pKD46/pKD78) in 10mL LB-Amp or LB-Cam overnight at 30°C
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Day 2
- Add 500uL of overnight culture to 30mL LB-Amp (or Cam).
- Grow on 30C shaker until OD600 = ~ 0.350 - 0.400, (takes ~2-3 hours)
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- Induce cultures with filtered 10% Arabinose, creating a final concentration of 0.1% Arabinose (300uL of 10% Arabinose into the 30mL of culture.
- Place back on 30°C shaker for 15 minutes, then transfer to 50mL falcon tube, and place on ice for 40 minutes, and start chilling centrifuge.
- Spin cultures @ 6000rpm x 5 min to pellet cells.
- Resuspend pellet in 30 mL cold 10% glycerol.
- Repeat spin and wash cyle twice more.
- After the final spin, pour out the glycerol as completely as possible without disturbing the pellet, and place the tube back in the bucket. Ideally, the rest of the residual liquid will be ~300uL, and therefore enough for ~6 electroporations. Vortex or swirl the tube to resuspend the pellet, or use a 1mL pipet to resuspend.
- Aliquot 50 uL cell suspension into cold eppendorf tubes.
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- Induce cultures with filtered 10% Arabinose, creating a final concentration of 0.1% Arabinose (300uL of 10% Arabinose into the 30mL of culture).
- Place back on 30°C shaker for 15 minutes, then transfer to 50mL falcon tube, and place on ice for 40 minutes. Start chilling centrifuge to 4C.
- Spin cultures @ 1800 x g for 15 min to pellet cells.
- Resuspend pellet in 30 mL cold water.
- Spin @ 3000 x g for 5 minutes, resuspend in cold water.
- Spin @ 3000 x g for 5 minutes, and resuspend in cold 10% glycerol.
- After this spin cycle, while pouring out the glycerol pay close attention to the pellet. It is likely somewhat loose/mushy at this point, so pour off the glycerol until the pellet starts to move, then briefly vortex to resuspend the entire pellet, and transfer the entire remaining volume to a cold 15mL tube. Remaining volume will likely be between 10 to 3 mL.
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- Rebalance centrifuge, spin @ 3000 x g for 5 minutes. Pour off all glycerol, pellet should be stable.
- Add back cold 10% glycerol for desired cell density, and number of elctroporations. Usually 300uL will yield a good cell density for ~6 electroporations. Resuspend pellet by pipetting with 1mL pipette.
- Aliquot 50 uL cell suspensions into cold eppendorf tubes.
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Transforming Cells
- Add DNA to cell aliquots, typically 50-200ng (be sure to desalt, or use very low volumes of DNA i.e., 1uL)
- Transfer cells + DNA to a chilled gap cuvette
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- Electroporate, recover immediately with 1mL SOC/SOB/or LB
- Transfer to a culture tube, place on 37°C shaker for 1.5 hrs
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- Electroporate, recover immediately with 1mL SOC/SOB/LB.
- Transfer to a culture tube with 10mL SOC/SOB/LB, place on 30°C shaker for 1.5 hrs.
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- Plate 100-200 uL of recovery onto selection plate (such as LB-Kan if using the Kan cassette from the keio collection). Leave the recovery culture on bench overnight, plate more next day if the initial plating was unsuccessful.
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- Grow the selection plate at 37°C overnight; Colonies may take a full 24 hours to appear
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- Grow the selection plate at 37°C overnight; Colonies may take a full 24 hours to appear.
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NOTE Recover at 30 deg if you intend on maintaining pKD46. Plasmid is usually lost at 37°C |
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> | Expected Results
The data below used 20ng of purified PCR product (900bp) targeting the kan cassette from the topB keio strain, and 50uL of lambda-ready cells. 3 electroporations per strain.
REL606
| Colonies on selective plate |
Colonies on control plate |
Dilution Factor |
Recombination Efficiency |
| 5 |
65 |
10^5 |
7.7E-8 |
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| 8 |
37 |
10^5 |
2.2E-7 |
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| 7 |
53 |
10^5 |
1.3E-7 |
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BW25113
| Colonies on selective plate |
Colonies on control plate |
Dilution Factor |
Recombination Efficiency |
| 1 |
135 |
10^5 |
7.4E-9 |
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| 1 |
152 |
10^5 |
6.6E-9 |
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| 3 |
125 |
10^5 |
2.4E-8 |
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| | References
- Datsenko, K.A. & Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645.
Contributors
- Originally from Erik Quandt (6/7/2011)
- Edited by Steven Sowa (6/2011)
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- Edited by -- Main.NeilGottel - 14 Jun 2012
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- Edited by -- Main.NeilGottel - 04 Sep 2012
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