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| | ==Making Ultracompetent Cells== | | ==Making Ultracompetent Cells== |
| - | *Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. DNA blotted on paper by the registry tends to be even harder. We have had much better luck with the following protocol from Inoue than the standard CaCl2 method: | + | *Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. DNA blotted on paper by the registry tends to be even harder. We have had a great experience with the following protocol from Inoue, getting near lawns of transformants from ligations: |
| | + | |
| | + | *Based on the OpenWetWare page: http://openwetware.org/wiki/Preparing_chemically_competent_cells_(Inoue) |
| | + | |
| | + | *Makes about 25mL, or enough for nearly 500 50 uL transformations |
| | | | |
| | ==Materials== | | ==Materials== |
| - | * Plate of cells streaked for single colonies | + | * Plate of cells streaked for single colonies. We used DH5alpha. |
| - | * [[SOB]] | + | * [http://openwetware.org/wiki/SOB SOB] |
| | * Ice | | * Ice |
| - | * [[TB buffer]] | + | * [http://openwetware.org/wiki/TB_buffer TB buffer] |
| | * DMSO | | * DMSO |
| | * Dry Ice (or liquid nitrogen) | | * Dry Ice (or liquid nitrogen) |
| | | | |
| | ==Glassware & equipment== | | ==Glassware & equipment== |
| - | * 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water) | + | * (3)1 liter Erlenmeyer flasks (no detergent residue- we rinsed well with pure water, then autoclaved 2/3 full of pure water, emptying just before use |
| - | * 220 ml conical centrifuge tubes BD 35 2075 | + | * (1) 250 mL Erlenmeyer flask |
| - | * Eppendorf 5410R refrigerated centrifuge with conical adapters | + | * 250 mL centrifuge bottles, autoclaved |
| | + | * Refrigerated centrifuge |
| | | | |
| | ==Preparation== | | ==Preparation== |
| - | # Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask. Save some medium as an OD blank. | + | ===Day 1=== |
| - | # Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours. | + | # Pour LB plates (no antibiotic) |
| | + | # In the afternoon, streak a plate of DH5alpha from frozen stock. (We usually just use a sterile 200 ul pipette tip, scrape some frozen cells off the top, melt them in a pool near an edge of the plate, return the still-frozen stock to the -80C freezer, and streak back and forth across the surface.) |
| | + | #Incubate plate inverted at 37C overnight (14-20 hours) |
| | + | # Make 1L of SOB medium in a bottle without detergent residue |
| | + | # Rinse out the clean flasks very well to remove detergent residue- (3) 1L, (1) 250 mL. Fill each 2/3 with pure water and autoclave. Keep the water in overnight. |
| | + | |
| | + | ===Day 2=== |
| | + | # At around 11 AM, pour the water out of the 250 mL flask and add 25mL SOB |
| | + | # Innoculate with one colony from the LB plate |
| | + | # Grow this starter culture at 37C on a 250 rpm shaker for 6-8 hours |
| | + | # At around 6 PM, pour the water out of your (3) 1L flasks and add 250mL of SOB to each. Label them 10, 4 and 2. |
| | + | # Innoculate these cultures with 10, 4, and 2 mL of the starter culture, respectively. |
| | + | # Incubate at room temperature (the lower the better, down to around 20C) at 100 rpm |
| | + | # Put 140 .5mL microcentrifuge tubes in a rack, and place them in a sealed bag in the -20 or -40 freezer overnight. |
| | + | # Label two pipet tip boxes or similar with the strain, your initials, the date, and either 150 uL or 100 uL. Store at -40C overnight |
| | + | |
| | + | ===Day 3=== |
| | + | |
| | + | |
| | + | rto an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours. |
| | # Prechill the centrifuge to 4 degrees | | # Prechill the centrifuge to 4 degrees |
| | # Remove from the incubator and place on ice for 10 minutes | | # Remove from the incubator and place on ice for 10 minutes |
Making Ultracompetent Cells
- Transformation can be a major roadblock in assembling DNA constructs. Ligations can be hard to transform, especially with large inserts. DNA blotted on paper by the registry tends to be even harder. We have had a great experience with the following protocol from Inoue, getting near lawns of transformants from ligations:
- Based on the OpenWetWare page: http://openwetware.org/wiki/Preparing_chemically_competent_cells_(Inoue)
- Makes about 25mL, or enough for nearly 500 50 uL transformations
Materials
- Plate of cells streaked for single colonies. We used DH5alpha.
- [http://openwetware.org/wiki/SOB SOB]
- Ice
- [http://openwetware.org/wiki/TB_buffer TB buffer]
- DMSO
- Dry Ice (or liquid nitrogen)
Glassware & equipment
- (3)1 liter Erlenmeyer flasks (no detergent residue- we rinsed well with pure water, then autoclaved 2/3 full of pure water, emptying just before use
- (1) 250 mL Erlenmeyer flask
- 250 mL centrifuge bottles, autoclaved
- Refrigerated centrifuge
Preparation
Day 1
- Pour LB plates (no antibiotic)
- In the afternoon, streak a plate of DH5alpha from frozen stock. (We usually just use a sterile 200 ul pipette tip, scrape some frozen cells off the top, melt them in a pool near an edge of the plate, return the still-frozen stock to the -80C freezer, and streak back and forth across the surface.)
- Incubate plate inverted at 37C overnight (14-20 hours)
- Make 1L of SOB medium in a bottle without detergent residue
- Rinse out the clean flasks very well to remove detergent residue- (3) 1L, (1) 250 mL. Fill each 2/3 with pure water and autoclave. Keep the water in overnight.
Day 2
- At around 11 AM, pour the water out of the 250 mL flask and add 25mL SOB
- Innoculate with one colony from the LB plate
- Grow this starter culture at 37C on a 250 rpm shaker for 6-8 hours
- At around 6 PM, pour the water out of your (3) 1L flasks and add 250mL of SOB to each. Label them 10, 4 and 2.
- Innoculate these cultures with 10, 4, and 2 mL of the starter culture, respectively.
- Incubate at room temperature (the lower the better, down to around 20C) at 100 rpm
- Put 140 .5mL microcentrifuge tubes in a rack, and place them in a sealed bag in the -20 or -40 freezer overnight.
- Label two pipet tip boxes or similar with the strain, your initials, the date, and either 150 uL or 100 uL. Store at -40C overnight
Day 3
rto an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
- Prechill the centrifuge to 4 degrees
- Remove from the incubator and place on ice for 10 minutes
- Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
- Drain the medium and resuspend each pellet first in 5 ml of ice cold TB. Add an additional 75 ml of cold TB buffer and resuspend.
- Place on ice for 10 minutes
- Spin down as above.
- While spinning, add 1.4 ml of DMSO to 18.6 ml of TB (7% DMSO mixture)
- Resuspend each pellet in 20 ml of cold TB-DMSO mixture
- Incubate on ice for 10 minutes
- Dispense cells into pre-chilled tubes
- Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
Thoughts on improvements
- "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
- They also control pH at 7.5, which may be a major issue
- Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
- Length of time on ice prior to transformation may make a big difference
- The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.
- Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
- My lab uses LB for instead of SOB media...it seems to work fine for them--mel 18:10, 14 June 2007 (EDT)
Related topics & references
Original protocol from Inoue et al. Inoue90 . Useful comments and speculation about reducing agents in Hengen96.
<biblio>
- Inoue90 pmid=2265755
- Hengen96 pmid=8851666
</biblio>
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