Team:Minnesota/Notebook
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<h1>Protocols: Standard techniques that we used in the wet lab</h1> | <h1>Protocols: Standard techniques that we used in the wet lab</h1> | ||
<h3>Bacterial Culture</h3> | <h3>Bacterial Culture</h3> |
Revision as of 14:25, 28 July 2009
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Protocols: Standard techniques that we used in the wet lab
Bacterial Culture
Sterile Technique
- Always work around a flame or in the hood
- Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping
- Sterilize metal instruments between uses by dipping in 100% ethanol and flaming
Bacterial Culture Maintenance
Culture cells:- At 36 degrees Celsius
- Shaking at 220 rpm
- At 10% total flask/tube volume
- In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x108cell/ml)
Bacterial Culture For Gene Expression Experiments
- Pick and individual colony from a plate and inoculate 2ml LB + amp media
- Incubate overnight at 37 C, shaking at 220 rpm
- Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture
- Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)
- Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture
- Continue cultures as described above in "bacterial culture maintenance" for 9 hrs
- Isolate cell samples from cultures at 3, 6, and 9 hour time points
- Remove 100ul sample aliquots from cultures
- Pellet samples at 5K rpm for 5 minutes
- Remove supernatant
- Wash cells with 1 ml chilled 1xPBS, pH 7.6
- Resuspend cells by vortexing
- Re-pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)
- Incubate at RT for 30 minutes
- Pellet cells at 5K rpm for 5 minutes
- Remove supernatant
- Resuspend cells in 1 ml 1xPBS
- Store samples at 4 C until analysis by flow cytometry
Transformation of Chemically Competent Cells
- Thaw cells and incubate transformant DNA in ice(~15 minutes)
- Combine 50 ul cells with ~3uL DNA and mix gently
- Incubate samples on ice for 15 minutes
- Heat shock cells in 42C water bath of 50 seconds
- Incubate samples on ice for 5 minutes
- Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm
- Transfer cells to a 2 ml microfuge tube
- Spin cells down at 6K rpm for 2 minutes
- Remove all but ~100uL supernatant media
- Resuspend cells gently in remaining media
- Plate cells on LB + ab plates
- Incubate plates overnight at 37C
DNA Work
Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)
- Pick and individual colony from a plate and inoculate 2ml LB + ab media
- Incubate culture overnight at 37C
- Transfer culture to 2 ml microfuge tube
- Spin cells down at 13K rpm for 2 min at RT and remove supernatant
- Resuspend cells in 250 ul Buffer P1 (stored at 4 C)
- Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue
- Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless
- Centrifuge sample at 13K rpm for 10 minutes
- Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind
- Centrifugre supernatant into column at 13K rpm for 1 minute
- Remove the flowthrough
- Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute
- Remove the flowthrough
- Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute
- Remove the flowthrough
- Spin out residual liquid at 13K rpm for 1 minute
- Place column in a fresh 1.5 ml microfuge tube
- Elute DNA; apply 40 ul Buffer EB to column, incubate at room temperature for 2 minutes and spin out of column at 13K rpm for one minute
DNA quantification
- Dilute DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul
- Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul
- Read absobance of blank and DNA sample at lambda = 260 and 280
- Calculate [DNA]; [DNA](ng/ul) = DF*A260*50
- Determine sample purity; pure DNA A260/A280 = 1.8
Polymerase Chain Reaction(PCR)
- Combine the following on ice:
- Thermocycle
- Repeat 25-35 cycles of segments 2-4
- Combine the following on ice:
- Incubate at 37C for 2-20 hours
- Heat inactive the enzyme at 65C for 15 minutes
- Combine the following on ice
- Incubate at 37C for 30 minutes
- Heat inactive enzyme at 65C for 15 minutes
- Combine the following on ice:
- Vector insert ratio should be about 1:3-- lower ratios may decrease insertion efficiency and higher ratios may lead to cancatamerization of inserts. Additionally, 100ng <= total DNA <= 500ng
- Incubate at 16C overnight or at RT for 30 minutes(overnight ligation is preferred)
- Heat inactivate enzyme at 65C for 15 minutes
- Add 5 volumes of Buffer PB1to 1 volume DNA and mix
- Apply DNA sample to QIA quick column
- Spin DNA into column at 13K rpm for 1 minute
- Discard flowthrough
- Wash DNA with 0.75 ml Buffer PE, spin through column at 13K rpm for 1 minute
- Discard flowthrough
- Spin residual liquid from column at 13K rpm for 1 minutes
- Elute DNA; apply 40ul Buffer EB to column, incubate at room temperature for 2 minutes before spinning DNA out of column at 13K rpm for 1 minute
- Make an agarose gel at 0.8<= gel density <= 1.5
- Add loading dye to samples (5 ul dye/50 ul sample)
- Load samples and a ladder (5 ul) into gel wells
- Run samples through gel (negative to positive) at 100 V for 40 minutes at room temperature
- Visualize DNA under UV light
- Excise gel piece containing DNA with a new razor
- Add three volumes of Buffer QG to 1 volume gel
- Incubate at 50C for 15 minutes, or until gel is solublized, mixing frequently
- Make sure dissolved solution is yellow
- If the DNA fragment is <500bp and >4kb, add 1 gel volume isopropanol to increase the yield
- Apply sample to QIAquick spin column in 700 ul aliquots
- Spin sample into column at 13K rpm for 1 minute
- Discard flowthrough
- Spin 0.5 ml Buffer QG through column at 13K rpm for 1 minute to solublize any remaining gel chunks
- Discard flowthrough
- Wash column with 0.75 ml Buffer PE, spinning through column at 13K rpm for 1 minute
- Discard flowthrough
- Spin out residual liquid from column at 13K rpm for 1 minute
- Elute DNA by applying 40-50ul Buffer EB to column, incubate at room temperature for 2 minutes, spin DNA out of the column at 13K rpm for 1 minute into a clean microfuge tube
- Combine the following in 0.5ml epindorf tube:
- Submit to BMGC for sequencing
Reagent | 1x(volume in ul) | [final] | |
---|---|---|---|
10x Thermo Pol Buffer | 5 | 1x | |
10mM dNTPs | 1 | 0.2mM | |
50mM MgCl2 | 2 | 2mM | |
10 uM F Primer | 2 | 0.4mM | |
10 uM R Primer | 2 | 0.4uM | |
H2O | Vol req for 50 ul total | - | |
DNA | Vol req for 30 ng <= mass <= 500ng | - | |
Taq polymerase | 0.5 | 2 U | |
Total | 50 ul | - |
Segment | Temperature(C) | Length | |
---|---|---|---|
1. Initial Denaturation | 94 | 3 minutes | |
2. Denaturation | 94 | 30 seconds | |
3. Annealing | ~55 | 30 seconds | |
4. Extension | 68 | 1 min/kb amplified | |
5. Final Extension | 68 | 5 minutes | |
6. Final Hold | 12 | Forever |
Restriction Digest
Reagent | 1x(volume in ul) | [final] | |
---|---|---|---|
10x NEB Buffer | 5 | 1x | |
BSA | 0.5 | 1x | |
H2O | Vol req for 50 ul total | - | |
DNA | Vol req for 0.5 ng <= mass <= 5 ug | - | |
Restriction Enzyme | 0.5 | 1 U | |
Total | 50 ul | - |
Vector Dephosphorylation
Reagent | 1x(volume in ul) | [final] | |
---|---|---|---|
10x AP Buffer | 5 | 1x | |
H2O | Vol req for 50 ul | - | |
DNA | Vol req for 0.5 ug <= mass <= 5ug | - | |
Antarctic Phosphate | 0.5 | 1 U |
DNA Fragment Ligation
Reagent | 1x(volume in ul) | [final] | |
---|---|---|---|
10x Ligase Buffer | 2 | 1x | |
H2O | Vol req for 20 ul | - | |
Vector DNA | 3 <= fmoles <= 30 | - | |
Insert DNA | 9 <= fmoles <=90 | - | |
T4 DNA Ligase | 1 | 1 U | |
Total | 20 | - |
DNA purification
Purify DNA (Using QIAquick PCR purification)
Separate DNA by size on an agarose gel
Purify DNA from Gel (Using QIAquick Gel Extraction Kit
Sequencing
Reagent | 1x(vol in ul) | [Final] | |
---|---|---|---|
10 uM seq primer | 1 | 10 pmol | |
DNA | Vol req for 300ng | 300 ng | |
H2O | Vol req for 12 uL | - | |
12 ul | - |