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/ul)
      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 16ul
      2. Add 2.5ul of NEBuffer 2.1
      3. Check here for buffer selection (depending on the enzyme)
      4. Add 0.5ul of BSA
      5. Add 0.5ul of EcoRI
      6. Add 0.5ul of PstI
      7. There should be a total volume of 20ul. Mix well and spin down briefly
      8. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
      9. Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
   3. Source
      1. iGEM http://parts.igem.org/Help:Protocols/Restriction_Digest


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


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. https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf


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. https://openwetware.org/wiki/Preparing_chemically_competent_cells


5. Making competent Lactococcus L. lactis NZ9000 cells
   1. Materials
      1. GM17
      2. SGM17 + glycine
      3. 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. https://openwetware.org/wiki/Lactococcus_transformation
   
   
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. http://parts.igem.org/Help:Protocols/Transformation


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 50-100µl and plate on a selection plate
      9. Incubate the plates at 30°C for 24 - 48 hours
   3. Source
      1. https://openwetware.org/wiki/Lactococcus_transformation


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. https://www.qiagen.com/us/resources/download.aspx?id=c73208eb-a83e-40c4-a9b6-ea5c4c94b9f4&lang=en


9. Quickchange PCR


10. 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 https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273


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. https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530


12. PCR cleanup, Gel extraction, minipreps was performed by using kits provided by Qiagen. Standard procedure was followed.


13. Media
    1. Lysogeny Broth (LB)
       1. Dissolve the following in 1L ddH2O
       2. 10g bacto-tryptone
       3. 5g bacto-yeast extract
       4. 10g NaCl (5g in some recipes)
       5. Adjust to pH 7.0 with NaOH
    2. M17
       1. Dissolve the following in 1L ddH2O
       2. 5.0 g Pancreatic Digest of Casein
       3. 5.0 g Soy Peptone
       4. 5.0 g Beef Extract
       5. 2.5 g Yeast Extract
       6. 0.5 g Ascorbic Acid
       7. 0.25 g Magnesium Sulfate
       8. 10.0 g Disodium-β-glycerophosphate
       9. 11.0 g Agar
    3. SOC
       1. Dissolve the following in 1L ddH2O
       2. 20g Bacto Tryptone
       3. 5g Bacto Yeast Extract
       4. 2ml of 5M NaCl
       5. 2.5ml of 1M KC
       6. 10ml of 1M MgCl2
       7. 10ml of 1M MgSO4
       8. 20ml of 1M glucose


14. Antibiotic
    1. Chloramphenicol
    2. Erythromycin
    3. Ampicillin