Protocols

1. Restriction Digestion
   1. Materials
      1. (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
      2. BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/μl)
      3. dH2O
      4. NEB Buffer 2
      5. BSA
      6. Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
   2. Procedure
      1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16 μl
      2. Add 2.5 μl of NEBuffer 2.1
      3. Check here for buffer selection (depending on the enzyme)
      4. Add 0.5 μl of BSA
      5. Add 0.5 μl of EcoRI
      6. Add 0.5 μl of PstI
      7. There should be a total volume of 20 μl. Mix well and spin down briefly
      8. Incubate the restriction digest at 37°C for 30 min
      9. If prevention of self-ligation of backbone is recommended: add 0.5 μl alkaline phosphatase and incubate for another 20 min 37°C.
      10. Incubate at 80°C for 20 min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
      11. Run a portion of the digest on a gel (8 μl, 100ng), to check that both plasmid backbone and part length are accurate.
   3. Source
      1. iGEM



2. Ligation
   1. Materials
      1. Digested backbone & inserts
      2. T4 DNA ligase
      3. T4 DNA ligase buffer
   2. Procedure
      1. Add 2 μl of digested plasmid backbone (25 ng)
      2. Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 μl)
      3. Add equimolar amount of XbaI PstI digested fragment (< 3 μl)
      4. Molar ratios of 1:1, 1:10, 1:20 are recommended
      5. Add 1 μl T4 DNA ligase buffer. Note: Do not use quick ligase
      6. Add 0.5 μl T4 DNA ligase
      7. Add water to 10 μl
      8. Ligate 16C/30 min, heat kill 80C/20 min
      9. Transform with 1-2 μl of product
   3. Source
      1. iGEM


3. Gibson assembly
   1. Materials
      1. Compatible Fragments
      2. Gibson Assembly Master Mix 2x
      3. Positive control (NEB)
   2. Procedure

      	2-3 Fragment Assembly	4-6 Fragment Assembly	Positive Control**
      Total Amount of Fragments	0.02–0.5 pmols* X μl	0.2–1 pmols* X μl	10 μl
      Gibson Assembly Master Mix (2X)	10 μl	10 μl	10 μl
      Deionized H2O	10-X μl	10-X μl	0
      Total Volume	20 μl***	20 μl***	20 μl

   3. Source
      1. NEB


4. Preparing competent E. coli DH5α cells
   1. Materials
      1. Plate or stock of E.coli DH5α cell
      2. LB
      3. TSS buffer
      4. Ice
   2. Procedure
      1. Grow 5ml overnight culture of cells in LB media, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask.
      2. In the morning You should aim to dilute the overnight culture by at least 1/100.
      3. Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
      4. Put Eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being cooled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
      5. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
      6. All subsequent steps should be carried out at 4°C and the cells should be kept on ice whenever possible.
      7. Centrifuge for 10 minutes at 3000 rpm and 4°C.
      8. Decant supernatant, remove leftover media by carefully pipetting
      9. Resuspend in TSS buffer (10% of original volume), vortex gently
      10. Add 100 μl aliquots to chilled Eppendorfs, flash freeze and store at – 80°C in 200 µl aliquots.
   3. Source
      1. Openwetware


5. Making competent Lactococcus L. lactis NZ9000 cells
   1. Materials
      1. M17
      2. GM17
      3. SGM17 + glycine
      4. Electroporation buffer
   2. Procedure
      1. Grow cells overnight in 25ml of GM17
      2. Add 1ml of overnight culture into 25ml SGM17 + glycine
      3. Grow for ~4 hours until OD600 ~ 0.7
      4. Chill culture on ice for 10 mins
      5. Centrifuge cells for 15 mins at 3000g
      6. Gently shake to resuspend pellet in 3ml Electroporation Buffer
      7. Centrifuge cells for 15 mins at 3000g
      8. Resuspend pellet in 3ml Electroporation Buffer
      9. Centrifuge cells for 15 mins at 3000g
      10. Resuspend pellet in 500 µl Electroporation Buffer
      11. Separate into 100 µl aliquots and store at -80°C until use.
   3. Source
      1. Openwetware
   
   
6. Transformation E.coli DH5α
   1. Materials
      1. Plasmid DNA
      2. Competent E. coli DH5α cells: 50 µl per transformation
      3. 2x SOC stock
      4. SOC salt stocks
      5. Sterile MQ
      6. LB agar selection plates: Two per transformation
      7. Eppendorf tubes
      8. Floater
      9. Ice
      10. 42°C water bath
      11. 37°C incubator: both shaker and stove
      12. Sterile spreader/glass beads
   2. Procedure (on ice)
      1. Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.
      2. Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes
      3. Pipet 1-100 ng of DNA as well as control into tubes and gently mix with tip
      4. incubate on ice for 30 min, tubes may be gently flicked, return to ice ASAP
      5. Meanwhile, for every transformation, add 2 µl of each SOC salt solution into 100 µl 2x SOC stock and ad to 200 µl with sterile MQ. Place the SOC medium on ice till use
      6. Heat shock tubes at 42°C for 30 seconds (precisely)
      7. Incubate on ice for 5 min
      8. Add 200 µl of SOC medium to each transformation
      9. Incubate at 37°C for 1 hours, shaker or rotor recommended
      10. Pipet 20 µl & 200 µl transformation mixture onto petri plates and spread with sterilized spreader or glass beads. Let the plates dry near the flame before placing them in the incubator
      11. Incubate plates upside down overnight (14-18hr) at 37°C
      12. Pick single colonies
      13. Perform Colony PCR to verify
      14. Grow cells & miniprep
      15. Calculate efficiency by counting colonies (expected value: 1.5x10^8 to 6x10^8 cfu/µg DNA)
   3. Source
      1. iGEM


7. Electrotransformation L. Lactis
   1. Materials
      1. Plasmid DNA (preferably without salts from buffers. These can be removed by incubating the DNA on a filter, which is floating on MQ for 10 minutes at room temperature
      2. Electrocompetent L. lactis cells: 50 µl per transformation
      3. Recovery medium
      4. Electroporation Cuvettes
      5. Electroporator
      6. SGM17 agar selection plates
      7. Eppendorf tubes
      8. Ice
      9. Eppendorf centrifuge
      10. 30°C incubator
      11. Sterile spreader/glass beads
   2. Procedure
      1. Mix all elements of the recovery medium (1 ml per transformation) and place it on ice together with the electroporation cuvettes
      2. Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.
      3. Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes
      4. Add at most 5 µl of plasmid DNA to the competent cells and incubate on ice for 10 minutes
      5. Dry the cuvette, electroporate at 2500 V and carefully add 950 µl recovery medium to the cells. Place the cuvette back on ice and incubate for 10 minutes
      6. Transfer the cell suspension with careful mixing into Eppendorf tubes and incubate for 2 hours at 30°C
      7. Plate 100 µl of the recovered cells onto a selection plate and spin down the remaining cells at 6000 rpm for 5 minutes
      8. Resuspend cell pellet into 50-100 µl and plate on a selection plate
      9. Incubate the plates at 30°C for 24 - 48 hours
   3. Source
      1. Openwetware


8. Colony PCR
   1. Materials
      1. 10X Standard Taq Reaction Buffer
      2. 10 mM dNTPs
      3. 10 µM Forward Primer
      4. 10 µM Reverse Primer
      5. Template DNA (colony resuspended in MQ / plasmid DNA)
      6. Taq DNA Polymerase
      7. Nuclease-free water
      8. PCR tubes
      9. Ice
      10. PCR tube rack
   2. Procedure
      1. Suspend a colony in 10 µl sterile MQ
      2. Prepare Mastermix for 10 reactions according to:

         Component	220 µl = 10 colonies	Final Concentration
         10X Standard Taq (Mg-free) Reaction Buffer	22 µl	1X
         25 mM MgCl2	13,2 µl	1.5 mM
         10 mM dNTPs	4,4 µl	200 µM
         10 µM pJET fw	4,4 µl	0.2 µM (0.05–1 µM, typically 0.1-0.5µM)
         10 µM pJET rv	4,4 µl	0.2 µM (0.05–1 µM, typically 0.1-0.5µM)
         Taq DNA Polymerase	1,1 µl	1.25 units/50 µl PCR
         Nuclease-free water	148,5 µl	-

      3. Put 19 µl of mastermix in each reaction tube & add 2 µl suspended colony mixture
      4. Add 2 µl plasmid DNA for positive control and 2 µl MQ for the negative control
      5. Place the tubes in the PCR machine (Taq program)
      6. Once the PCR is done, mix 10 µl of PCR product with 2 µl 6X purple gel loading dye and run it on a gel for 50 minutes at 130 Volts
      7. If the correct products are present in the gel samples, inoculate overnight cultures from the original plates.
      8. Mix 5 ml LB with appropriate antibiotic. Scoop a colony from the plate and drop the tip into the medium. Incubate the tube at 37°C overnight to let the culture grow
   3. Source
      1. Qiagen


9. Taq PCR
   1. Materials according to table
   2. Procedure
      1. Mix according to table

         Component	25 μl reaction	50 μl reaction	Final Concentration
         10X Standard Taq Reaction Buffer	2.5 μl	5 μ	1X
         10 mM dNTPs	0.5 µl	1 μl	200 µM
         10 µM Forward Primer	0.5 µl	1 μl	0.2 µM (0.05–1 µM)
         10 µM Reverse Primer	0.5 µl	1 μl	0.2 µM (0.05–1 µM)
         Template DNA	variable	variable	1,000 ng
         Taq DNA Polymerase	0.125 µl	0.25 µl	1.25 units/50 µl PCR
         Nuclease-free water	to 25 µl	to 50 µl	-

      2. PCR cycler conditions

         Step	Temperature	Time
         Initial Denaturation	95°C	30 sec
         30 cycles	95°C	15-30 sec
         30 cycles	45°C-68°C	15-60 sec
         30 cycles	68°C	1 min/ kb
         Final extension	68°C	5 min
         Hold	4-10°C	-

   3. Source
      1. NEB


10. HotStar HiFidelity PCR
    1. Materials according to table
    2. Procedure
       1. Mix according to table

          Component	Volume/reaction products	Final Concentration
          5x HotStar HiFidelity PCR Buffer (contains dNTPs)	10 μl	1X
          5x Q-Solution	10 µl	1x
          10 µM Forward Primer	5 µl	1 µM
          10 µM Reverse Primer	5 µl	1 µM)
          Template DNA	variable	0.1-50 ng
          Hot Star HiFidelity DNA Polymerase	1 µl for <2k fragments, 2 µl for 2-5 kb	2,5 units, 5 units
          Nuclease-free water	to 50 µl	-

       2. PCR cycler conditions

       3. Step	Temperature	Time
          Initial Denaturation	95°C	5 min
          30 cycles	94°C	15 sec
          30 cycles	50°C-68°C (extract 5°C from typical Tm)	1 min
          30 cycles	72°C for <2kb, 68°C for 2-5 kb	1 min/kb, 2 min/kb
          Final extension	72°C	10 min
          Hold	4-10°C	-

       4. Source
          1. Qiagen
    
    
11. Phusion PCR
    1. Materials according to table
    2. Procedure

       Component	20 μl reaction	50 μl reaction	Final Concentration
       5X Phusion HF/ GC Buffer	4 μl	10 μl	1X
       10 mM dNTPs	0.4 µl	1 μl	200 µM
       10 µM Forward Primer	1 µl	2,5 μl	0.5 µM (0.05–1 µM)
       10 µM Reverse Primer	1 µl	2,5 μl	0.5 µM (0.05–1 µM)
       Template DNA	variable	variable	<250 ng
       Phusion DNA Polymerase	0.2 µl	0.5 µl	1. units/50 µl PCR
       Nuclease-free water	to 20 µl	to 50 µl	-

    3. PCR cycler conditions

    4. Step	Temperature	Time
       Initial Denaturation	98°C	30 sec
       30 cycles	98°C	5-10 sec
       30 cycles	45°C-72°C	10-30 sec
       30 cycles	72°C	15-30 sec/ kb
       Final extension	72°C	5-10 min
       Hold	4-10°C	-

    5. Source
       1. NEB


12. PCR cleanup
1. Materials
   1. QIAquick PCR Purification Kit
   2. Eppendorf tubes
   3. Eppendorf centrifuge
2. Procedure
   1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction ad mix. If the color of the mixture is orange or volet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
   2. Place a QIAquick column in a provided 2 ml collection tube
   3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s. Discard the flow-through and place the QIAquick column back in the same tube
   4. To wash, add 0.75 ml of Buffer PE to the QIAquick column and centrifuge for 30-60 s. Discard flow-through and place the QIAquick column back in the same tube
   5. Centrigue the QIAquick column once more in the provided 2 ml colletion tube for 1 min to remove residual wash buffer
   6. Place each QIAquick column in a clean Eppendorf tube
   7. To elute DNA, add 50 µl Buffer EB (10 mM Tris-Hcl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
   8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
3. Source
   1. Qiagen



13. Gel extraction
1. Materials
   1. QIAquick Gel Extraction Kit
   2. 50°C water bath
   3. Eppendorf tubes
   4. Eppendorf centrifuge
2. Procedure
   1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
   2. Weigh te gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl). The maxiumum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Vuffer QG
   3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow
   4. Add 1 gel volume isopropanol to the sample and mix
   5. Place a QIAquick spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >80 µl, load and spin again
   6. To wash, add 750 µl Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
   7. Place QIAquick column into a clean 1.5 ml microcentrifuge tube
   8. To elute DNA, add 50 µl Buffer EB (10 mM Tris-Hcl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
   9. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel
3. Source
   1. Qiagen



14. Plasmid isolation
1. Materials
   1. QIAprep Spin Miniprep Kit
   2. Eppendorf tubes
   3. Eppendorf centrifuge
2. Procedure
   1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
   2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
   3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
   4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
   5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
   6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30–60 s and discard the flow-through.
   7. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
   8. Centrifuge for 1 min to remove residual wash buffer.
   9. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
3. Source
   1. Qiagen



15. Phenol-Chloroform DNA extraction
1. Materials
   1. Phenol:chloroform:isoamyl alchohol (25:24:1)
   2. Mili-Q water
   3. Eppendorf centrifuge
   4. Chemical fume hood
   5. Nitrile gloves
   6. Eppendorf tubes
   7. At least 100 μl sample (if you have 20 μl ligation, add 80 μl Mili-Q)
   8. SpeedVac
2. Procedure
   1. Warning: phenol can cause severe burns to the eyes and the skin. Chloroform is a volatile liquid that affects the central nervous system and a suspected human carcinogenic. It is very volatile and can cause damage by inhalation, skin and eyes absorption, and ingestion. Therefore, perform the whole procedure in a chemical fume hood and wear nitrile gloves (if accidental contact occurs, remove and discard contaminated gloves immediately)
   2. Add one volume of phenol:chloroform:isoamyl alcohol to the sample.
   3. Vortex vigorously for 1 minute.
   4. Spin at 10.000g for 5 minutes. (to separate organic and aqueous phases)
   5. Place the top aqueous solution in a new eppendorf tube and discard the organic bottom phase.
   6. Note: Be careful do not pick up any of the phenol:chloroform:isoamyl alcohol phase.
   7. Repeat the above steps two times.
   8. Now, you can concentrate the DNA sample by using SpeedVac or ethanol precipitation.
3. Source
   1. We received this protocol from our supervisor Patricia.



16. Ethanol DNA precipitation
1. Materials
   1. 100% cold ethanol
   2. 70% cold ethanol
   3. Eppendorf centrifuge
   4. Sodium acetate (3M) (pH 5.2)
   5. MiliQ water or elution buffer
   6. Eppendorf tubes
   7. At least 100 μl sample (if you have 20 μl ligation, add 80 μl Mili-Q)
   8. SpeedVac
   9. Optional: glycogen
2. Procedure
   1. Add 0.1x volume of sodium acetate and mix (no vortex)
   2. Optional trick: add 1 μl of glycogen. Glycogen will co-precipate with the DNA and therefore, it will form a visible pellet and incease the recovery of DNA. It van be useful when you have small quantities of DNA.
   3. Add 2.5x volume of 100% cold ethanol and mix (no vortex).
   4. Incubate at -80 °C for at least 2 hours.
   5. Spin at top speed for 15 minutes at 4°C.
   6. Remove the supernatant carefully without touching the pellet.
   7. Spin for some seconds and remove again the excess of supernatant.
   8. Wash by adding 150 μl of 70% cold ethanol by carefully sliding the ethanol along the wall of the tube.
   9. Spin at top speed for 5 minutes at 4°C.
   10. Repeat the ethanol wash from step 8 twice.
   11. Dry with SpeedVac.
   12. Resuspend in the appropriate volume of MiliQ water or elution buffer.
3. Source
   1. We received this protocol from our supervisor Patricia.


17. Antibiotic
    1. E. coli
    1. Ampicillin: 100 μg/mL
    2. Chloramphenicol: 25 μg/mL
    3. Tetracycline: 10 μg/mL
    2. L. lactis
    1. Chloramphenicol: 5 μg/mL
    2. Erythromycin: 1 μg/mL
    
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