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JUDGING FORM ⇗

Protocols

Golden Gate Plasmid

1. Set up Amp. Plates (250 mL batch)

  • 8.9g Easy Mix
  • per 250 ml of water
  • add 0.5 ml of AMP to autoclaved mix (cooled, now room temp)
  • pour mix into plates and let dry

    • 2. Make 10mM Tris-HCl

    3. Evaluate how many pmol/fmol are in each DNA tube

  • 3.9 pmol = plasmid4 + scfv1
  • 4.9 pmol = linker region 1
  • 2.75 pmol = scfv1 + arac2
  • 4.3 pmol = linker region 2
  • 4.9 pmol = acrc2 + plasmid 1
  • 2.5 pmol = plasmid 1
  • 1.11 pmol = plasmid 2
  • 1.2 pmol = plasmid 3
  • 0.92 pmol = plasmid 4

    • 4. Bring up DNA to appropriate concentration (40fmol/1uL) using 10mM Tris-HCl

  • Add 97.5 μl to plasmid4 + scfv1
  • Add 122.5 μl to linker region 1
  • Add 68.8 μl to arac1 + scfv2
  • Add 107.5 μl to linker region 2
  • Add 122.5 μl to acrc2 + plasmid 1
  • Add 62.5 μl to plasmid 1
  • Add 27.8 μl to plasmid 2
  • Add 30.0 μl to plasmid 3
  • Add 23.0 μl to plasmid 4

    • 5. Assemble

  • 11.5 μl Nuclease-free H2O
  • 2 μl of T4 DNA Ligase Buffer (10X)
  • Add 0.5μl of each fragment 1
  • Add 0.5μl of each fragment 2
  • Add 0.5μl of each fragment 3
  • Add 0.5μl of each fragment 4
  • Add 0.5μl of each fragment 5
  • Add 0.5μl of each fragment 6
  • Add 0.5μl of each fragment 7
  • Add 0.5μl of each fragment 8
  • Add 0.5μl of each fragment 9
  • 2 μl NEB Golden Gate Enzyme Mix
  • *** total volume should be 20 μl

    • 6. Thermocycle/PCR

  • For 5-10 inserts: (37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min
  • For 11+ inserts: (37°C, 5 min → 16°C, 5 min) x 120 → 60°C, 5 min
  • Going to 16°C is optional, and may create more mismatches

    • 7. PCR Purification

    1. Add 100μl of Buffer PB and all the PCR liquid into a QIAquick spin column
    2. Centrifuge for 30-60sec
    3. Discard flow through
    4. Add 750μl buffer PE
    5. Centrifuge for 30-60sec
    6. Discard flow through
    7. Centrifuge again for 30-60 secs
    8. Discard flow
    9. Get new 1.5ml microcentrifuge tube and label it GG and date
    10. Add 50 μl water
    11. Centrifuge for 1min and put in freezer

    8. Transform (into E.Coli cells)

  • Keep everything on ice constantly
  • Warm up plates and SOC to 37
  • Make sure cells are somewhat thawed
    1. Add 2μl of DNA to microcentrifuge tube
    2. Add 25μl of E. coli cells to mc. tube
    3. Stir the liquid in the mc. tube with the pipet
    4. Pull all 27μl out of the tube
    5. Drop in between the two slits at the bottom of the corvette
    6. Perform electroporation ASAP, at 1.8 kV
    7. Record time constant (want to to be around 4.7)
    8. Add 960μl of the SOC broth into the box
    9. Pipet up and down a few times to mix
    10. Pull all the broth back out and put back into the tube
    11. Incubate at 37 for 1hr
    12. Stir it a bit (invert it several times and flick it)
    13. Plate 50μl per plate

    1. From primer powder make 100uM/L solution of primer

    2. Make 10uM/L stock solution of primer

  • 45 uL water
  • 5 uL 100 uM/L primer solution

    • 3a. If using DreamTaq Green PCR Master Mix

  • 25 uL of Master Mix
  • 0.1-1 uM FWD Primer (.5 uL of stock)
  • 0.1-1 uM REV Primer (.5 uL of stock)
  • 10 pg-1 ug Template DNA (0.5 uL)
  • Up to 50 uL Nuclease free H2O

    • 3b. If no Master Mix

  • 5uL of 10x standard Taq buffer
  • 1.5ul of 50 uM MgCl2
  • 1uL of 10 uM dNTPs
  • 1 uL of FWD primer (from 10uM stock!)
  • 1 uL of REV primer (from 10uM stock!)
  • ~1.25uL of template DNA or from colonies
  • 0.25uL of Taq DNA polymerase
  • 39uL of dH2O (Up to 50 uL of water)

    • 4a. If using DNA set Thermocycler to

  • Hold
  • 94 C - 3 min
  • 25-40 cycles
  • 94 C - 30 sec
  • Tm-5 C - 30 sec
  • 68 C - 1 min
  • Hold
  • 68 C - 5-15 min
  • 4 C - infinity /li>

    4b. If using colony as template DNA set Thermocycler to

  • Hold
  • 95 C - 5 min
  • 25-40 cycles
  • 95 C - 30 sec
  • 58 C - 60 sec
  • 68 C - 1 min
  • Hold
  • 68 C - 5 min
  • 4 C - infinity

    • EcoRI Digest

  • 8 uL DNA (150 ng/uL) = 0.15ug
  • 1 uL EcoRI
  • 1 uL CutSmart
  • 10 uL total volume
  • Incubate 70 mins at 37 C
  • Heat inactivate for 20 mins at 65 C

    • Pst1 Digest

  • 8 uL DNA (150 ng/uL) = 0.15ug
  • 1 uL Pst1
  • 1 uL CutSmart
  • 10 uL volume total
  • Incubate at 37 C for at least one hour
  • Heat inactivate at 80 C for 20 min

    • Use the QIAprep Miniprep Kit

  • Pellet 1-5ml of bacterial overnight culture by centrifugation at >8000rpm for 3min at room temperature
  • Resuspend the pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube
  • Add 250μl BufferP2 and mix throughly by inverting the tubes until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5min
  • Add 350μl Buffer N3 and mix immediatly and thoroughly by inverting the tube 4-6 times. If using the LyseBlue reagent the solution will turn colorless
  • Centrifuge for 10min at 13,000 RPM in a tabletop centrifuge
  • Apply 800μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting
  • Centrifuge for 30-60sec and discard flow through at 10,000 RPM
  • Wash the QIAprep 2.0 spin column by adding 0.5ml Buffer PB
  • Centrifuge for 30-60sec. Discard flow through
  • Wash the QIAprep 2.0 spin column by adding 0.75ml Buffer PE
  • Centrifuge for 30-60sec. Discard flow through
  • Centrifuge for 1 min to remove residual wash buffer
  • Place the QIAprep 2.0 column in a clean 1.5ml microcentrifuge tube.
  • Add 40μl of water and let stand for 1-2 min
  • Centrifuge for 1 min

    • Ingredients:

  • 10g Tryptone
  • 5g Yeast Extract
  • 10g Sodium Chloride

    • Steps:

  • Dissolve the ingredients in 1L water
  • Portion into flasks and cover with aluminum foil (fill flask to half its volume)
  • Autoclave for 20-30 minutes on LIQUID cycle

    • Steps:

  • Prepare microcentrifuge at room temperature. Place NEB® 10-beta/Stable Outgrowth Medium in a 37°C water bath. Pre-warm plates at 37°C for 1 hour.
  • Place electroporation cuvettes and microcentrifuge tubes on ice
  • Positive control for transformation: dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using ddH2O.
  • Thaw NEB 10-beta Electrocompetent cells on ice (about 10 min) and mix cells by flicking gently.
  • Transfer 25 μl of the cells to chilled microcentrifuge tubes and add 1 uL of DNA
  • Transfer the cell/DNA mix into chilled cuvette (without introducing bubbles)
  • Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 1.8 kV. The typical time constant is 4.8 to 5.1 milliseconds.
  • Immediately add 975 uL of 37°C NEB 10-beta/Stable Outgrowth Medium to the cuvette
  • Gently mix up and down twice and then transfer to the microcentrifuge tube
  • Shake (250 rpm) or rotate at 37°C for 1 hour
  • Dilute the cells (as appropriate); spread 100-200 μl cells onto a pre-warmed plate.
  • Incubate plates overnight at 37°C.

    • Lorem Ipsum!

    Steps:

  • Excise the DNA band from the agarose el with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.
  • Weigh the gel slice in a colorless tube. Add buffer QX1 according to DNA fragment size; 3 volumes with 2 volumes of water for >4kb.
  • Resuspend QIAEX II by vortexing for 30 sec. Add QIAEX II to the sample and mix: use 10 ul QIAEX II for ≤2 ug DNA; 30 ul for 2-10 ug DNA; andan additional 30 ul for each additional 10 ug DNA.
  • Incubate at 50 degrees for 10 min to solubilize the agarose and bind the DNA. Mix by vortexing every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 ul 3M sodium acetate, pH 5.0, and mix. The color should turn yellow. The incubation should then be continued for at least 5 min.
  • Centrifuge the sample for 30 sec and carefully remove supernatant with a pipet.
  • Wash the pellet with 500 ul buffer QX1. Resuspend the pellet by vortexing.
  • Centrifuge the sample for 30 sec and remove all traces of supernatant with a pipet. This step removes residual agarose contaminants.
  • Wash the pellet twice with 500 ul Buffer PE. Resuspend peet by vortexing.
  • Centrifuge the sample for 30 sec and carefully remove all traces of supernatant with a pipet. This step removes residual salt contaminants.
  • Air-dry the pellet for 10-15 min or until the pellet becomes white. If 30 ul QIAEX II suspension is used, air dry the pellet approx. 30 min.
  • To elute DNA, add 20 ul of water and resuspend the pellet by vortexing. Incubate according to the DNA fragment size: 5 min at 50 degrees for 4-10 kb.
  • Centrifuge for 30 sec, and carefully pipet the supernatant into a clean tube. The supernatant now contains the purified DNA.

    • FOR C2527H CELLS

    Perform these steps in the tube provided:

  • Thaw a tube of BL21 cells on ice for 10 minutes until last bit of ice disappears
  • Add 1-5 uL containing 1pg - 100 ng of plasmid DNA to the cell mixture
  • Carefully flick the tube 4-5 times to mix the cells and DNA (do not vortex)
  • Place the mixture on ice for 30 minutes (do not mix)
  • Heat shock at exactly 42 C for exactly 10 seconds (do not mix)
  • Place on ice for 5 minutes (do not mix)
  • Pipette 950 uL of room temperature SOC into the mixture
  • Place at 37 C for 60 minutes - shake vigorously (250 rpm) or rotate

    • These steps do not need to be in the tube:

  • Warm plates to 37 C
  • Mix the cells thoroughly by flicking the tube and inverting
  • Spread 20 uL, 50 uL, and 100 uL onto plates and incubate overnight at 37 C
  • Alternatively, incubate at 30 C for 24-36 hours or at 25 C for 48 hours

    • DAY ONE

  • Streak out frozen glycerol stock of bacterial cells (Top 10, DHα, etc.) onto an LB plate (no antibiotics)
  • Grow plate overnight at 37°C

    • DAY TWO

    1. Autoclave:

  • 2L ddH2O
  • 100 mL of 10% v/v glycerol
  • 1 L LB (or your preferred media)
  • 4 centrifuge bottles and caps
  • 2 50mL small centrifuge bottles
  • Lots of microcentrifuge tubes

    • 2. Chill overnight at 4°C:

  • ddH2O
  • 10% glycerol
  • Centrifuge rotor

    • 3. Prepare starter culture of cells:

  • Select a single colony of E. coli from fresh LB plate and inoculate a 10mL stater culture of LB (or your prepared media)
  • Grow culture at 37°C in shaker overnight
  • Notes: You can subsitute other media like SOB, 2xYT, etc for LB if preferred. All glassware should be detergent free. Trace detergent residue reduces competency.

    • DAY THREE

    1.

  • Inoculate 1 L of LB media at 37°C with 10mL starter culture and grow in 37°C shaker.
  • Measure the OD600 every hour, then every 15-20 mins when the OD gets above 0.2 AND STOP when OD reaches 0.35-0.40

    • 2.

  • When the OD600 reaches 0.35-0.4, immediately put the cells on ice.
  • Chill the culture for 20-30 mins, swirling occasionally to ensure even cooling
  • Place centrifuge bottles on ice

    • IMPORTANT NOTES:

  • Carefully check and monitor often OD once the OD reaches 0.2
  • DO NOT let OD get above 0.4
  • It usually takes about 3 hours to reach an OD of 0.35 when using a 10mL starter culture.
  • Keep the cells at 4°C for the remainder of the procedure (any bottles or solutions that the cells come into contact with must be pre-chilled to 4°C

    • 3.

  • (SPIN 1) Split the 1 L culture into four parts by pouring about 250mL into ice cold centrifuge bottles.
  • Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C

    • 4.

  • Decant the supernatant and resuspend each pellet in 200mL of ice cold ddH2O

    • 5.

  • (SPIN 2) harvest the cels by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C

    • 6.

  • Decant the supernatant and resuspend each pellet in 100mL of ice cold ddH2O

    • 7.

  • (SPIN 3) Combine resuspensions into 2 centrifuge bottles (so each contains about 200mL of cell suspension)
  • Harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C
  • Rinse two 50mL concical tubes with ddH2O and chill on ice

    • 8.

  • Decant the supernatant and resuspend each pellet in 40mL of ice cold 10% glycerol
  • Transfer each suspension to a 50mL concical tube

    • 9.

  • (SPIN 4)harvest the cells by centrifugation at 1000g (~2400 rpm in the Beckman JA-rotor) for 20 mins at 4°C
  • Start putting 1.5mL microcentrifuge tubes on ice if not already chilled

    • 10.

  • Carefully aspirate the supernatant with a sterile pasteur pipette (pellets lose adherance in 10% gylcerol)
  • Resuspend each pellet in 1mL of ice cold 10% gylcerol by gently swirling
  • The final OD600 of the resuspended cells should be 200-250

    • 11.

  • Aliquot into sterile 1.5mL microcentrifuge tubes and snap freeze with liquid nitrogen
  • Store frozen cells in -80°C freezer

    • THE PROTOCOL

    1. PCR cleanup backbone

    2. 30 uL of DNA (cleaned)

  • 5 uL 10X Cutsmart buffer
  • 1 uL BsaI
  • 1 uL DpnI

    • 3. incubate for 4 hr at 37 C

    4. gel extract using 1.0% gel and ~90V (if needed)

  • 1 uL Quick CIP
  • 15 ul of DNA
  • 2 uL cutsmart buffer
  • 3 uL of dH2O ~ up to 20 ul

    • 5. incubate for 1 hr at 37 C

    6. heat inactivate at 80 C for 4 mins