Team:UTDallas/Protocols
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{{Template:UTDallasMenu}} | {{Template:UTDallasMenu}} | ||
<span style="color:#EDE8E8"> | <span style="color:#EDE8E8"> | ||
+ | __NOEDITSECTION__ | ||
=Protocols= | =Protocols= | ||
+ | ===Ligation Protocol=== | ||
+ | *Determine insert to vector ratios | ||
+ | *Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector) | ||
+ | *In a PCR tube add the following: | ||
+ | **50ng of vector | ||
+ | **Amount of insert based on ratios (calculated in second step) | ||
+ | **2uL of buffer | ||
+ | **2uL of DNA ligase | ||
+ | **Amount of water to bring total volume to 20uL | ||
+ | *Incubate overnight at 14<sup>o</sup>C | ||
+ | |||
+ | Note: We used T4 DNA ligase and buffer from NEB | ||
+ | |||
+ | ===Gel Purification Protocol (from QIAquick Gel Extraction Kit)=== | ||
+ | *Excise DNA fragment from the agarose gel with a clean, sharp scalpel | ||
+ | *Weigh the gel slice in a microcentrifuge tube. | ||
+ | *Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL) | ||
+ | *Incubate at 50<sup>o</sup>C for 10 min (until the gel slice has completely dissolved) | ||
+ | *After the gel slice has dissolved completely, check that the color of the mixture is yellow | ||
+ | *Apply the sample to a QIAquick column, and centrifuge for 1 min | ||
+ | **Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again. | ||
+ | *Discard flow-through and place QIAquick column back in the same collection tube | ||
+ | *To wash, add 750uL of Buffer PE to column and centrifuge for 1 min. | ||
+ | *Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm | ||
+ | *Place QIAquick column into a clean 1.5 mL microcentrifuge tube | ||
+ | *To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min. | ||
+ | |||
+ | ===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 | ||
+ | |||
+ | ===Digestion Protocol=== | ||
+ | *Using a microcentrifuge tube add the following: | ||
+ | **~3000-5000 ng of DNA | ||
+ | **10uL Buffer 4 | ||
+ | **10uL BSA | ||
+ | **5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes) | ||
+ | **Amount of H<sub>2</sub>O needed to make final volume 100uL | ||
+ | *Incubate at 37<sup>o</sup>C for 1hr and 30min | ||
+ | |||
+ | Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4. | ||
+ | |||
+ | ===Preparing LB+Appropriate Antibiotic Protocol=== | ||
+ | *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 4<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 | ||
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** Let sit under flames until gel solidifies | ** Let sit under flames until gel solidifies | ||
** Replace lids on plates | ** Replace lids on plates | ||
- | *Store upside down at | + | *Store upside down at 4<sup>o</sup>C |
===Preparing Competent Cells Protocol=== | ===Preparing Competent Cells Protocol=== | ||
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*For long term storage, use 0.1M CaCl<sub>2</sub> in 15% glycerol at step 6 and store cells at -800<sup>o</sup>C | *For long term storage, use 0.1M CaCl<sub>2</sub> in 15% glycerol at step 6 and store cells at -800<sup>o</sup>C | ||
- | 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 4<sup>o</sup>C |
+ | |||
+ | ===Miniprep Protocol (from QIAprep Spin Miniprep Kit)=== | ||
+ | *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. | ||
+ | |||
+ | ===Preparing Glycerol Stock Protocol=== | ||
+ | *Add 150 µL of 50% glycerol to 350 µL of cells | ||
+ | *Place in -80<sup>o</sup>C 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 42<sup>o</sup>C for 45 sec | ||
+ | *Incubate the cells on ice for 2 minutes | ||
+ | *Under flame, add 450 µL SOC broth | ||
+ | *Incubate at 37<sup>o</sup>C for 1 hour while rotating or shaking at 300rpm | ||
+ | *Spread cells on appropriate antibiotic LB plates (usually 100 µL) | ||
+ | *Incubate at 37<sup>o</sup>C 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 | ||
</span> | </span> |
Latest revision as of 19:31, 20 October 2010
Protocols
Ligation Protocol
- Determine insert to vector ratios
- Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)
- In a PCR tube add the following:
- 50ng of vector
- Amount of insert based on ratios (calculated in second step)
- 2uL of buffer
- 2uL of DNA ligase
- Amount of water to bring total volume to 20uL
- Incubate overnight at 14oC
Note: We used T4 DNA ligase and buffer from NEB
Gel Purification Protocol (from QIAquick Gel Extraction Kit)
- Excise DNA fragment from the agarose gel with a clean, sharp scalpel
- Weigh the gel slice in a microcentrifuge tube.
- Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)
- Incubate at 50oC for 10 min (until the gel slice has completely dissolved)
- After the gel slice has dissolved completely, check that the color of the mixture is yellow
- Apply the sample to a QIAquick column, and centrifuge for 1 min
- Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
- Discard flow-through and place QIAquick column back in the same collection tube
- To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.
- Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm
- Place QIAquick column into a clean 1.5 mL microcentrifuge tube
- To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.
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
Digestion Protocol
- Using a microcentrifuge tube add the following:
- ~3000-5000 ng of DNA
- 10uL Buffer 4
- 10uL BSA
- 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)
- Amount of H2O needed to make final volume 100uL
- Incubate at 37oC for 1hr and 30min
Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.
Preparing LB+Appropriate Antibiotic Protocol
- 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 4oC
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 4oC
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 4oC
Miniprep Protocol (from QIAprep Spin Miniprep Kit)
- 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.
Preparing 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