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Team:UTDallas/Protocols
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Note: Harvest cells at 5000 rpm for 10 minutes at 40<sup>o</sup>C | Note: Harvest cells at 5000 rpm for 10 minutes at 40<sup>o</sup>C | ||
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| + | ===Miniprep Protocol=== | ||
| + | *Harvest cells at 5400g 10 minutes 40<sup>o</sup>C (possibly program 1) | ||
| + | *Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube | ||
| + | *Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times | ||
| + | *Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times | ||
| + | *Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge | ||
| + | *Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting | ||
| + | *Centrifuge for 30-60 seconds. Discard the flow-through | ||
| + | *Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds | ||
| + | *Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer | ||
| + | *To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute. | ||
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Revision as of 16:21, 28 July 2010
Protocols
Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)
- 300 mL DI H2O + 11 g LB agar
- Autoclave
- Put control thermometer in H2O (from the sink)
- Select vented container mode (Do Not Change Program)
- Mix well after autoclaving; let cool to 50oC
- Add antibiotic (50 to 100 µg/mL) (15 mg total)
- Weigh on paper
- Add to 0.5 mL DI H2O
- Add to LB mixture when cool enough
- Plate
- Under flame open lids of all plates
- Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
- Let sit under flames until gel solidifies
- Replace lids on plates
- Store upside down at 40oC
Preparing Competent Cells Protocol
- Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37oC and 200-300 rpm
- Inoculate 0.25 mL of the overnight strain into 25 mL of LB
- Shake at 37oC until the OD650 is 0.6-0.7
- Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2
- Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2
- Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2
- Leave on ice for 30 minutes
- For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800oC
Note: Harvest cells at 5000 rpm for 10 minutes at 40oC
Miniprep Protocol
- Harvest cells at 5400g 10 minutes 40oC (possibly program 1)
- Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube
- Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times
- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
- Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge
- Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting
- Centrifuge for 30-60 seconds. Discard the flow-through
- Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds
- Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer
- To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.
