Team:UTDallas/Protocols
From 2010.igem.org
(Difference between revisions)
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**Run at 130V for 30min-1hr | **Run at 130V for 30min-1hr | ||
+ | ===Prepare LB+Appropriate Antibiotic=== | ||
+ | *200 mL LB broth | ||
+ | *Autoclave | ||
+ | ** Put control thermometer in H<sub>2</sub>O (from the sink) | ||
+ | ** Select vented container mode (Do Not Change Program) | ||
+ | *Let cool to 50<sup>o</sup>C | ||
+ | *Add antibiotic (50-100 ug/mL) (10 mg total) | ||
+ | ** Weigh on paper | ||
+ | ** Add to 0.5 mL DI H<sub>2</sub>O | ||
+ | ** Add to LB mixture when cool enough | ||
+ | *Store at 40<sup>o</sup>C | ||
+ | |||
===Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)=== | ===Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)=== | ||
*300 mL DI H2O + 11 g LB agar | *300 mL DI H2O + 11 g LB agar |
Revision as of 20:23, 19 October 2010
Protocols
Gel Electrophoresis Protocol
- Making a 1% agarose gel
- 100mL 1X TBE buffer
- 1g agarose
- microwave until agarose dissolves
- let mixture cool
- when cool add 8-10uL ethidium bromide
- stir gently, let cool
- pour into plate with comb already in place
- let harden
- Using the gel
- Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)
- Load 2uL of DNA ladder into the gel
- Load DNA into the gel
- Run at 130V for 30min-1hr
Prepare LB+Appropriate Antibiotic
- 200 mL LB broth
- Autoclave
- Put control thermometer in H2O (from the sink)
- Select vented container mode (Do Not Change Program)
- Let cool to 50oC
- Add antibiotic (50-100 ug/mL) (10 mg total)
- Weigh on paper
- Add to 0.5 mL DI H2O
- Add to LB mixture when cool enough
- Store at 40oC
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.
Prepare Glycerol Stock Protocol
- Add 150 µL of 50% glycerol to 350 µL of cells
- Place in -80oC freezer
Transformation Protocol
- With a pipette tip, punch a hole through the foil cover of the DNA plate
- Add 10 µL of DI water
- Thaw competent cells on ice
- Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes
- Incubate the cells on ice for 30 minutes
- Heat shock the cells at 42oC for 45 sec
- Incubate the cells on ice for 2 minutes
- Under flame, add 450 µL SOC broth
- Incubate at 37oC for 1 hour while rotating or shaking at 300rpm
- Spread cells on appropriate antibiotic LB plates (usually 100 µL)
- Incubate at 37oC for 18-24 hours
- Take a colony, put in 3 mL of LB + appropriate antibiotic
- Use resulting culture to miniprep DNA and make your own glycerol stock