Team:Minnesota/Notebook

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Protocols: Standard techniques that we used in the wet lab

Bacterial Culture

Sterile Technique

  1. Always work around a flame or in the hood
  2. Flame the mouth and cap of any bottle, flask or tube upon uncapping and recapping
  3. Sterilize metal instruments between uses by dipping in 100% ethanol and flaming

Bacterial Culture Maintenance

Culture cells:
  1. At 36 degrees Celsius
  2. Shaking at 220 rpm
  3. At 10% total flask/tube volume
  4. In mid-log phase(0.1 < OD600 <= 0.4) (with OD600 = 1 ->8.8x108cell/ml)

Bacterial Culture For Gene Expression Experiments

  1. Pick and individual colony from a plate and inoculate 2ml LB + amp media
  2. Incubate overnight at 37 C, shaking at 220 rpm
  3. Inoculate fresh media with overnight culture such that new culture has 2.5% inoculum; this is the secondary culture
  4. Incubate at 37 C shaking at 220 rpm until OD600 = 0.4 (~2 hrs)
  5. Inoculate 4 ml LB + amp + inducer (aTc or IPTG) with 100ul secondary culture
  6. Continue cultures as described above in "bacterial culture maintenance" for 9 hrs
  7. Isolate cell samples from cultures at 3, 6, and 9 hour time points
  8. Remove 100ul sample aliquots from cultures
  9. Pellet samples at 5K rpm for 5 minutes
  10. Remove supernatant
  11. Wash cells with 1 ml chilled 1xPBS, pH 7.6
  12. Resuspend cells by vortexing
  13. Re-pellet cells at 5K rpm for 5 minutes
  14. Remove supernatant
  15. Fix cells; resuspend cells in 1 ml 4% PFA (in PBS)
  16. Incubate at RT for 30 minutes
  17. Pellet cells at 5K rpm for 5 minutes
  18. Remove supernatant
  19. Resuspend cells in 1 ml 1xPBS
  20. Store samples at 4 C until analysis by flow cytometry

Transformation of Chemically Competent Cells

  1. Thaw cells and incubate transformant DNA in ice(~15 minutes)
  2. Combine 50 ul cells with ~3uL DNA and mix gently
  3. Incubate samples on ice for 15 minutes
  4. Heat shock cells in 42C water bath of 50 seconds
  5. Incubate samples on ice for 5 minutes
  6. Recover cells in 0.5 ml SOC media, shaking at 37C for 1 hour at 220 rpm
  7. Transfer cells to a 2 ml microfuge tube
  8. Spin cells down at 6K rpm for 2 minutes
  9. Remove all but ~100uL supernatant media
  10. Resuspend cells gently in remaining media
  11. Plate cells on LB + ab plates
  12. Incubate plates overnight at 37C



DNA Work


Plasmid Prep from cultures (using QIAprep Spin Miniprep Kit)

  1. Pick and individual colony from a plate and inoculate 2ml LB + ab media
  2. Incubate culture overnight at 37C
  3. Transfer culture to 2 ml microfuge tube
  4. Spin cells down at 13K rpm for 2 min at RT and remove supernatant
  5. Resuspend cells in 250 ul Buffer P1 (stored at 4 C)
  6. Add 250 ul Buffer P2 and mix thoroughly by inverting-- the solution should turn blue
  7. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting-- the solution should turn colorless
  8. Centrifuge sample at 13K rpm for 10 minutes
  9. Transfer supernatant to a fresh QIAprep spin column, leaving cell debris pellet behind
  10. Centrifugre supernatant into column at 13K rpm for 1 minute
  11. Remove the flowthrough
  12. Wash column with 0.5 ml Buffer PB; apply to column and spin through at 13K rpm for 1 minute
  13. Remove the flowthrough
  14. Wash column with 0.75 ml Buffer PE; apply to column and spin through at 13K rpm for 1 minute
  15. Remove the flowthrough
  16. Spin out residual liquid at 13K rpm for 1 minute
  17. Place column in a fresh 1.5 ml microfuge tube
  18. 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

  1. Dilute DNA as appropriate in water (1<= DF <=1/100) to a total volume of 50 ul
  2. Similarly dilute blank DNA buffer solution with water to a total volume of 50 ul
  3. Read absobance of blank and DNA sample at lambda = 260 and 280
  4. Calculate [DNA]; [DNA](ng/ul) = DF*A260*50
  5. Determine sample purity; pure DNA A260/A280 = 1.8

Polymerase Chain Reaction(PCR)

  1. Combine the following on ice:
  2. Reagent1x(volume in ul)[final]
    10x Thermo Pol Buffer51x
    10mM dNTPs10.2mM
    50mM MgCl222mM
    10 uM F Primer20.4mM
    10 uM R Primer20.4uM
    H2OVol req for 50 ul total-
    DNAVol req for 30 ng <= mass <= 500ng-
    Taq polymerase0.52 U
    Total50 ul-
  3. Thermocycle
  4. SegmentTemperature(C)Length
    1. Initial Denaturation943 minutes
    2. Denaturation9430 seconds
    3. Annealing~5530 seconds
    4. Extension681 min/kb amplified
    5. Final Extension685 minutes
    6. Final Hold12Forever
  5. Repeat 25-35 cycles of segments 2-4

Restriction Digest

  1. Combine the following on ice:
  2. Reagent1x(volume in ul)[final]
    10x NEB Buffer51x
    BSA0.51x
    H2OVol req for 50 ul total-
    DNAVol req for 0.5 ng <= mass <= 5 ug-
    Restriction Enzyme0.51 U
    Total50 ul-
  3. Incubate at 37C for 2-20 hours
  4. Heat inactive the enzyme at 65C for 15 minutes

Vector Dephosphorylation

  1. Combine the following on ice
  2. Reagent1x(volume in ul)[final]
    10x AP Buffer51x
    H2OVol req for 50 ul-
    DNAVol req for 0.5 ug <= mass <= 5ug-
    Antarctic Phosphate0.51 U
  3. Incubate at 37C for 30 minutes
  4. Heat inactive enzyme at 65C for 15 minutes

DNA Fragment Ligation

  1. Combine the following on ice:
  2. Reagent1x(volume in ul)[final]
    10x Ligase Buffer21x
    H2OVol req for 20 ul-
    Vector DNA3 <= fmoles <= 30-
    Insert DNA9 <= fmoles <=90-
    T4 DNA Ligase11 U
    Total20-
  3. 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
  4. Incubate at 16C overnight or at RT for 30 minutes(overnight ligation is preferred)
  5. Heat inactivate enzyme at 65C for 15 minutes

DNA purification

Purify DNA (Using QIAquick PCR purification)
  1. Add 5 volumes of Buffer PB1to 1 volume DNA and mix
  2. Apply DNA sample to QIA quick column
  3. Spin DNA into column at 13K rpm for 1 minute
  4. Discard flowthrough
  5. Wash DNA with 0.75 ml Buffer PE, spin through column at 13K rpm for 1 minute
  6. Discard flowthrough
  7. Spin residual liquid from column at 13K rpm for 1 minutes
  8. 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
Separate DNA by size on an agarose gel
  1. Make an agarose gel at 0.8<= gel density <= 1.5
  2. Add loading dye to samples (5 ul dye/50 ul sample)
  3. Load samples and a ladder (5 ul) into gel wells
  4. Run samples through gel (negative to positive) at 100 V for 40 minutes at room temperature
  5. Visualize DNA under UV light
Purify DNA from Gel (Using QIAquick Gel Extraction Kit
  1. Excise gel piece containing DNA with a new razor
  2. Add three volumes of Buffer QG to 1 volume gel
  3. Incubate at 50C for 15 minutes, or until gel is solublized, mixing frequently
  4. Make sure dissolved solution is yellow
  5. If the DNA fragment is <500bp and >4kb, add 1 gel volume isopropanol to increase the yield
  6. Apply sample to QIAquick spin column in 700 ul aliquots
  7. Spin sample into column at 13K rpm for 1 minute
  8. Discard flowthrough
  9. Spin 0.5 ml Buffer QG through column at 13K rpm for 1 minute to solublize any remaining gel chunks
  10. Discard flowthrough
  11. Wash column with 0.75 ml Buffer PE, spinning through column at 13K rpm for 1 minute
  12. Discard flowthrough
  13. Spin out residual liquid from column at 13K rpm for 1 minute
  14. 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


Sequencing

  1. Combine the following in 0.5ml epindorf tube:
  2. Reagent1x(vol in ul)[Final]
    10 uM seq primer110 pmol
    DNAVol req for 300ng300 ng
    H2OVol req for 12 uL-
    12 ul-
  3. Submit to BMGC for sequencing


Our Google Calendar

This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. You can scroll over each event to see a detailed description of what we did.