Swartz:Protocols/Maxiprep: Difference between revisions
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m (New page: https://docs.google.com/document/d/1woT8L647962azzQERKb_4_GdmsoBTWKpRtyVVSj2OTE/edit Qiagen DNA plasmid MaxiPrep Day 1: Inoculate 5 mL of selective LB broth from glycerol stock or plate ...) |
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Qiagen DNA plasmid MaxiPrep | Qiagen DNA plasmid MaxiPrep | ||
Day 1: | *Day 1: | ||
Inoculate 5 mL of selective LB broth from glycerol stock or plate colony | **Inoculate 5 mL of selective LB broth from glycerol stock or plate colony | ||
Grow at 37 °C, ~8 h with shaking | **Grow at 37 °C, ~8 h with shaking | ||
Autoclave 1L LB media | **Autoclave 1L LB media | ||
When cooled, add appropriate antibiotic | **When cooled, add appropriate antibiotic | ||
Add entire contents of 5mL starter culture to 1L media | **Add entire contents of 5mL starter culture to 1L media | ||
Grow at 37 °C, 12-16 h overnight with shaking | **Grow at 37 °C, 12-16 h overnight with shaking | ||
Day 2: | *Day 2: | ||
Dispense culture in 1L bottles and harvest in centrifuge at 6,500 rpm, 30 min | **Dispense culture in 1L bottles and harvest in centrifuge at 6,500 rpm, 30 min | ||
While culture is spinning, put buffer P3 on ice | **While culture is spinning, put buffer P3 on ice | ||
Resuspend the pellet with 40 mL of buffer P1 using the hand homogenizer | **Resuspend the pellet with 40 mL of buffer P1 using the hand homogenizer | ||
Add 40 mL of buffer P2 | **Add 40 mL of buffer P2 | ||
Mix thoroughly by inerting vigourously | **Mix thoroughly by inerting vigourously | ||
Incubate at room temperature for no longer than 5 min | **Incubate at room temperature for no longer than 5 min | ||
Add 40 mL of chilled buffer P3 | **Add 40 mL of chilled buffer P3 | ||
Incubate on ice, 20 min | **Incubate on ice, 20 min | ||
Spin down lysates at ~20,000g, 30 min, 4 °C | **Spin down lysates at ~20,000g, 30 min, 4 °C | ||
While spinning, prepare 2 Qiagen-500 tips per L of culture by adding 10 mL buffer QBT | **While spinning, prepare 2 Qiagen-500 tips per L of culture by adding 10 mL buffer QBT | ||
Remove supernatant into clean bottle by pipette | **Remove supernatant into clean bottle by pipette | ||
Filter through Qiagen filter (0.2-0.4 µm filters can also be used) | **Filter through Qiagen filter (0.2-0.4 µm filters can also be used) | ||
Load on columns by gravity flow | **Load on columns by gravity flow | ||
Wash columns 2x with 30 mL Wash Buffer | **Wash columns 2x with 30 mL Wash Buffer | ||
While loading and washing columns, place 25 mL isopropanol per L of culture in -30 °C freezer | **While loading and washing columns, place 25 mL isopropanol per L of culture in -30 °C freezer | ||
Elute with 15 mL buffer QF into clean 50 mL Falcon tube | **Elute with 15 mL buffer QF into clean 50 mL Falcon tube | ||
Precipitate DNA with 10.5 mL cold isopropanol | **Precipitate DNA with 10.5 mL cold isopropanol | ||
Centrifuge at ~20,000g, 30 min, 4 °C | **Centrifuge at ~20,000g, 30 min, 4 °C | ||
Pour of supernatant | **Pour of supernatant | ||
Be careful not to disturb DNA pellet | **Be careful not to disturb DNA pellet | ||
Wash the pellet with 0.5 mL of 70% ethanol | **Wash the pellet with 0.5 mL of 70% ethanol | ||
Transfer to 1.5 mL Eppendorf with transfer pipettes | **Transfer to 1.5 mL Eppendorf with transfer pipettes | ||
Centrifuge at 12,000 rpm, 20 min | **Centrifuge at 12,000 rpm, 20 min | ||
Pipette off ethanol | **Pipette off ethanol | ||
Allow pellet to dry either overnight on benchtop or 30-45 min in centrivap | **Allow pellet to dry either overnight on benchtop or 30-45 min in centrivap | ||
Resuspend pellet in 100-200 µl water | **Resuspend pellet in 100-200 µl water | ||
Flick tubes to speed up resuspension | **Flick tubes to speed up resuspension | ||
Allow to resuspend on benchtop for several hours or overnight | **Allow to resuspend on benchtop for several hours or overnight | ||
Check concentrations on spectrophotometer by measuring absorbance at 260 and 280 nm | **Check concentrations on spectrophotometer by measuring absorbance at 260 and 280 nm | ||
Revision as of 23:33, 14 May 2012
https://docs.google.com/document/d/1woT8L647962azzQERKb_4_GdmsoBTWKpRtyVVSj2OTE/edit
Qiagen DNA plasmid MaxiPrep
- Day 1:
- Inoculate 5 mL of selective LB broth from glycerol stock or plate colony
- Grow at 37 °C, ~8 h with shaking
- Autoclave 1L LB media
- When cooled, add appropriate antibiotic
- Add entire contents of 5mL starter culture to 1L media
- Grow at 37 °C, 12-16 h overnight with shaking
- Day 2:
- Dispense culture in 1L bottles and harvest in centrifuge at 6,500 rpm, 30 min
- While culture is spinning, put buffer P3 on ice
- Resuspend the pellet with 40 mL of buffer P1 using the hand homogenizer
- Add 40 mL of buffer P2
- Mix thoroughly by inerting vigourously
- Incubate at room temperature for no longer than 5 min
- Add 40 mL of chilled buffer P3
- Incubate on ice, 20 min
- Spin down lysates at ~20,000g, 30 min, 4 °C
- While spinning, prepare 2 Qiagen-500 tips per L of culture by adding 10 mL buffer QBT
- Remove supernatant into clean bottle by pipette
- Filter through Qiagen filter (0.2-0.4 µm filters can also be used)
- Load on columns by gravity flow
- Wash columns 2x with 30 mL Wash Buffer
- While loading and washing columns, place 25 mL isopropanol per L of culture in -30 °C freezer
- Elute with 15 mL buffer QF into clean 50 mL Falcon tube
- Precipitate DNA with 10.5 mL cold isopropanol
- Centrifuge at ~20,000g, 30 min, 4 °C
- Pour of supernatant
- Be careful not to disturb DNA pellet
- Wash the pellet with 0.5 mL of 70% ethanol
- Transfer to 1.5 mL Eppendorf with transfer pipettes
- Centrifuge at 12,000 rpm, 20 min
- Pipette off ethanol
- Allow pellet to dry either overnight on benchtop or 30-45 min in centrivap
- Resuspend pellet in 100-200 µl water
- Flick tubes to speed up resuspension
- Allow to resuspend on benchtop for several hours or overnight
- Check concentrations on spectrophotometer by measuring absorbance at 260 and 280 nm
