Team:ULV-LC-CV/Notebook

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Protocols


iGEM Transformation Protocol

1. Pre-chill microcentrifuge tube and thaw competent cells on ice.

2. Add 50 uL of competent cells into the chilled microcentrifuge tube.

3. Add 1 uL of DNA into the microcentrifuge tube.

4. Incubate on ice for 30 minutes.

5. Heat shock at 42 degrees Celsius for 1 minute.

6. Incubate cells on ice for 5 minutes.

7. Add 200 uL of SOC media into the microcentrifuge tube.

8. Incubate at 37 degrees Celsius (and shake if possible) for 1-2 hours.

9. Pipette 200 uL of cells onto LB plate with appropriate resistance.

10. Incubate plate overnight at 37 degrees Celsius.

Mini-prep

1. Transfer 1.5ml bacteria overnight cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000 rpm for 3 minutes at room temperature (15-25C)

2. Resuspend pelleted bacterial cells in 250l buffer PI

3. Add 250l buffer P2 and mix by inverting 4-6 times or until clear

4. Add 350l Buffer N3 and mix by inverting

5. Centrifuge for 10 minutes at 1300rpm

6. Pipette the suspected from step 5 to the QIAprep spin column. Centrifuge for 30-60 sec and discard the flow through

7. Wash the QIAprep spin column by adding 750l buffer PE. Centrifuge for 30-60 sec and discard the flow through

8. Place the QIAprep column in a cleans 1.5ml microcentrifuge tube. Add 50l Buffer EB to the center of the QIAprep spin column let it stand for 1minute and centrifuge for 1 minute

NEBioBrick Assembly Protocol

Digestion:

1. Thaw all enzymes need and aliquot appropriate amounts.

2. Pipette the amount of nuclease free water needed to make 500 ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50 uL to get the amount of water needed)

3. Add appropriate amount of DNA into the nuclease free water.

4. Add 3 uL of NEB Buffer to each tube.

5. Add 1 uL of the first enzyme to each tube. (E for upstream, X for downstream, E for vector)

6. Add 1 uL of the second enzyme to each tube. (S for upstream, P for downstream, P for vector)

7. Agitate each solution to mix well.

8. Incubate upstream and downstream parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.

9. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.

Ligation:

10. Thaw 10x T4 DNA ligase reaction buffer at room temperature. Mix well.

11. Add 11 uL of nuclease free water into a microcentrifuge tube.

12. Add 2 uL of each parts (upstream, downstream, and vector) into the tube with water.

13. Add 2 uL of 10X T4 DNA Ligase Buffer into the tube.

14. Add 1 uL of T4 DNA Ligase.

15. Incubate at room temperature for 10 minutes.

16. Incubate at 80 degrees Celsius to denature the enzymes and stop the ligation process.

17. Store in a -20 degrees Celsius freezer, or transform ligated DNA.

QIAEX II Gel Extraction Kit Protocol

1. Cut DNA band from the agarose gel with a clean, sharp razor blade. Weigh a 1.7mL centrifuge tube.

2. Add the DNA gel slice to weighed tube. Reweigh and subtract from original tube to find weight of DNA slice.

3. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100bp; 3 volumes for 100bp-4kb; 3 volumes with 2 volumes of water for >4kb; 6 volumes when using >2% agarose gels.

4. Resuspend QIAEX II by vortexing for 30s. Add QIAX II to the tube with the sample and vortex: Use 10µL of QIAEX II for ≤ 2µg DNA; 30µL for 2-10µg DNA; and an additional 30µL for each additional 10µg DNA.

5. Incubate at 50°C for 10min vortexing every 2min to melt agarose and bind DNA. Keep an eye on color change. If so adjust accordingly.

6. Centrifuge the sample for 30s and carefully remove supernatant.

7. Wash the pellet with 500µL Buffer QX1. Resuspend by vortexing. Centrifuge for 30s and remove supernatant.

8. Wash the pellet twice with 500µL Buffer PE. Resuspend pellet by vortexing. Centrifuge for 30s and remove supernatant.

9. Air-dry the pellet until it becomes white. Do not vacuum dry.

10. To elute DNA, add 20µL nuclease free water and resuspend the pellet by vortexing.* Incubate according to fragment size: 5min at room temp. for ≤4kb; 5min at 50°C for 4-10kb; 10min at 50°C for > 10kb.

        *Fragments larger than 10kb resuspend by flicking and inverting tube as vortexing can cause shearing.

11. Centrifuge for 30s and pipet supernatant into clean tube. This is your purified DNA.

12. Repeat steps 9 and 10 to combine eluates and increase yield.


Media recipes

SOB

  • All together in one bottle with screw cap add the following
  1. 2% Tryptone (20g per 1000ml)
  2. 0.5% Yeast Extract (5g per 1000ml)
  3. 10mM NaCl (0.5g per 1000ml)
  4. 2.5mM KCl (10ml of 250mM KCl (1.68 KCl in 100ml DiH2O) or 0.18 per 1000ml)
  5. Adjust pH to 7
  6. Autoclave for 15 min
  • Once cool, add strike MgCl2 enough so that it is 10mM

SOC

  • Same as SOB except add 20mM glucose
  1. Add 20ml of sterile 1M glucose per 1000ml
    To make 1M glucose, dissolve 18g of glucose (dextrose) in 100ml DiH2O. (Note: Filter Sterilize)

LB Media

  • 25g of LB Broth per 1000ml
  • If no LB Broth is available add the following:
    1. 10g Tryptone
    2. 5g yeast extract
    3. 10g NaCl
    • pH 7
    • Autoclave

    LB Agar Plates

    • Add 15 g agar to 1000mL LB media
    • Add 1uL of antibiotic per 1mLl
    • When cool to touch, poor 20ml into each plate in a sterile field

    Buffers


    Miniprep Buffers

    Re-suspension buffer (equivelent of Qiagen Buffer P1)
    • Tris – HCl-50mM
    • EDTA – 10mM
    • RNase A- 100g/ml
    • HCl – final pH8 (Note: store RNase A @ -20 degrees C, aliquot buffer and add at time of use, do not autoclave)

    Lysis buffer
    • NaOH – 200mM
    • SDS – 1% (w/v) (Note: do not autoclave SDS, use sterile filter)

    Neutralizeation Buffer (equivelent of Qiagen Biffer PB)
    • GU-HCl – 4.2 M
    • KOAc – 0.9 M
    • HOAc – final pH 4.2

    Column Wash/Blinding Buffer (equivelent of Qiagen Buffer PB)

    • GU – 5.0 M
    • Isoproponol – 30% (v/v)

    Column Wash Buffer (equivelent of Qiagen Biffer PE)
    • Tris-HCl – 10mM
    • Ethanol – 80% (v/v)
    • HCl – final pH 7.5

    Mini-column Recycling buffer(Equilibrium buffer (equivalent of Qiagen Buffer QBT))
    • NaCl – 750mM
    • MOPS – 50mM Isopropanol – 15% (v/v)
      • Triton X-100 – 0.15% (v/v) (Note: do not autoclave MOPS

    Protein Purification Buffer (NID Extraction Buffer)
    • EDTA – 20-50 mM
    • Tris – HCl – 50mM
    • NH4Cl -0.75 M
    • Triton X-100 – 0.5% (v/v)
    • Sucrose – 5% (w/v)
    • Lysozyme - 100g/ml
    • RNase A - 25g/m l
    • HCL – final pH 8 (Note: Store RNase A and Lysozyme @ -20 degrees Celcius. Aliquot buffer and add at time of use, do not autoclave)