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Our Protocols
From 2008.igem.org
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Same as for SOB above, but add 20mM glucose | Same as for SOB above, but add 20mM glucose | ||
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| + | |||
| + | ---- | ||
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| + | '''4.) Making Cells Electrocompetant''' | ||
| + | |||
| + | |||
| + | Time: ~1 hour, plus relevant cultures grown overnight | ||
| + | |||
| + | Required: | ||
| + | *25ml of overnight cultures | ||
| + | *500ml LB Medium | ||
| + | *Ice cold centrifuge flasks of relevant size | ||
| + | *dH20 | ||
| + | *Ice cold 1.5ml eppendorf tubes | ||
| + | |||
| + | |||
| + | |||
| + | ''Method'' | ||
| + | |||
| + | 1. Inoculate two aliquots of 500 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the | ||
| + | overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/minute in a rotary | ||
| + | shaker) | ||
| + | |||
| + | 2. Measure the OD600 of the growing bacterial cultures every 20 minutes. | ||
| + | |||
| + | 3. When the OD600 of the cultures reaches 0.4, rapidly transfer the flasks to an ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next | ||
| + | step, place the centrifuge bottles in an ice-water bath. | ||
| + | |||
| + | 4. Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g for 15 minutes at 4°C | ||
| + | |||
| + | 5. Decant the supernatant and resuspend the cell pellet in 500 ml of ice-cold pure H2O. | ||
| + | |||
| + | 6. Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. | ||
| + | |||
| + | 7. Decant the supernatant and resuspend the cell pellet in 250 ml of ice-cold 10% glycerol. | ||
| + | |||
| + | 8. Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C. | ||
| + | |||
| + | 9. Decant the supernatant and resuspend the pellet in 10 ml of ice-cold 10% glycerol. | ||
| + | |||
| + | 10. Harvest cells by centrifugation at 1000g for 20 minutes at 4°C. | ||
| + | |||
| + | 11. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any | ||
| + | remaining drops of buffer if you can. | ||
| + | |||
| + | 12. Resuspend the pellet in 1 ml of ice-cold GYT medium. | ||
| + | |||
| + | 13. Measure the OD600 of a 1:100 dilution of the cell suspension. Dilute the cell suspension to a | ||
| + | concentration of 2x10^10 to 3x10^10 cells/ml (1.0 OD600 = approx. 2.5x108 cells/ml) with ice-cold GYT | ||
| + | medium. | ||
| + | |||
| + | 14. Store the cells at -70°C until required. For storage, dispense 40-μl aliquots of the cell suspension into sterile, ice-cold 0.5ml microfuge tubes, drop into a bath of liquid nitrogen, and transfer to a -70°C freezer. | ||
Revision as of 21:16, 27 October 2008
1.) Making Agarose Gel (standard 1%)
Time: ~1 hour
Required:
- Gel tank, with combs, casting tray and relevant power supply
- Agarose powder
- TAE buffer, 1x
- Ethidium bromide
Method
1. The gel tank we normally use holds 150ml, therefore, take 150ml of 1x TAE Buffer
2. The gel is supposed to be 10% agarose, therefore we need to add 1.5g agarose powder
3. Combine the two in a chemical screw-cap bottle, and, leaving the lid slightly loose
4. Cook on high heat in the microwave for 2 mins, or until ALL the agarose has dissolved (it may stick to the bottom, swirling may be required. *CAUTION* very hot liquid)
5. If it starts to overboil, pause the microwave, allow to calm down, and continue.
6. When all the agarose has dissolved, remove carefully! Its very hot! Oven gloves may be required.
7. Add 10 µl of ethidium bromide per 100ml of buffer. Swirl to mix. Warning! Ethidium bromide is carcinogenic and therefore very dangerous!
8. Leave until touchably cool before pouring into gel tank. Gently bathe bottle in cold water to speed up the process if necessary.
9. Make sure combs are in place in tank, and casting tray is secured on all sides, then pour in the liquid.
10. Leave until cool (~30 mins)
2.) PCR Purification (Using QIAGEN Kit)
Time: ~1 hour
Required:
- PCR Purification kit ideally. Instructions and reagents usually provided
Method
1. Add 5 volumes of PBI buffer (provided) for every 1 volume of PCR product in an eppendorf tube. Make sure one eppendorf is large enough – you may need more.
2. Put mixture in a QIAGEN column (purple) and into a 2ml collection tube (clear)(both provided). Remember, these columns only hold 700µl! Again you may need to repeat steps 3 and 4 more than once.
3. Centrifuge at 10000-13000g for 30-60seconds
4. ‘Flow through’ will be found in the collection tube: discard. The DNA has stuck to the column membrane. Repeat if you had more than 700µl of PCR+PBI buffer to start with
5. The wash DNA of impurities further, add 0.75ml (750µl) of PE buffer (provided)(make sure the ethanol HAS been added to it! You have to do this yourself, but previous users of the kit may have done so and should have labelled the bottle so).
6. Centrifuge for 1 min, and discard flow through.
7. Centrifuge for a further 1min, as not all the PE buffer from the last step could fit in the collection tube.
8. Discard flow through and put column into where you want your eluate (usually 1.5ml eppendorfs)
9. To elute DNA, add 50µl of EB buffer (provided). This ‘unsticks’ the DNA allowing it to be washed through.
10. Centrifuge for 1 min
11. KEEP the flow through! This has your DNA in.
1.) Making SOB Medium
Time: ~20 mins, plus autoclaving time
Required:
EITHER
- SOB powder, in which case make up as instructions
OR, per litre
- 950ml dH20
- 20g Tryptone
- 5g Yeast Extract
- 0.5g NaCl
- 10ml of 250nM KCl
- 5ml of 2M MgCl2
- ~0.2ml 5M NaOH (may be required to adjust PH)
Method
1.) Combine dH2O, tryptone, yeats and NaCl, and shake until combined
2.) Add KCl
3.) Adjust to PH 7.0 with the NaOH
4.) Autoclave (20 mins liquid cycle)
5.) Add the MgCl2 just before use
4.) Making SOC Medium
Method
Same as for SOB above, but add 20mM glucose
4.) Making Cells Electrocompetant
Time: ~1 hour, plus relevant cultures grown overnight
Required:
- 25ml of overnight cultures
- 500ml LB Medium
- Ice cold centrifuge flasks of relevant size
- dH20
- Ice cold 1.5ml eppendorf tubes
Method
1. Inoculate two aliquots of 500 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/minute in a rotary shaker)
2. Measure the OD600 of the growing bacterial cultures every 20 minutes.
3. When the OD600 of the cultures reaches 0.4, rapidly transfer the flasks to an ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in an ice-water bath.
4. Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g for 15 minutes at 4°C
5. Decant the supernatant and resuspend the cell pellet in 500 ml of ice-cold pure H2O.
6. Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C.
7. Decant the supernatant and resuspend the cell pellet in 250 ml of ice-cold 10% glycerol.
8. Harvest the cells by centrifugation at 1000g for 20 minutes at 4°C.
9. Decant the supernatant and resuspend the pellet in 10 ml of ice-cold 10% glycerol.
10. Harvest cells by centrifugation at 1000g for 20 minutes at 4°C.
11. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer if you can.
12. Resuspend the pellet in 1 ml of ice-cold GYT medium.
13. Measure the OD600 of a 1:100 dilution of the cell suspension. Dilute the cell suspension to a concentration of 2x10^10 to 3x10^10 cells/ml (1.0 OD600 = approx. 2.5x108 cells/ml) with ice-cold GYT medium.
14. Store the cells at -70°C until required. For storage, dispense 40-μl aliquots of the cell suspension into sterile, ice-cold 0.5ml microfuge tubes, drop into a bath of liquid nitrogen, and transfer to a -70°C freezer.
