Team:Groningen/Protocols

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• Restriction digestion
• Ligation
• Gibson assembly
• Preparing cells
• Lactis cells
• Transformation E.coli
• Electrotransformation Lactis
• Colony PCR
• Quickchange PCR
• Taq PCR
• PCR cleanup
• Media
• Antibiotic

PROTOCOLS

Protocols

1. Restriction Digestion
   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
   1. 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.
   2. 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. 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. PEG 800
      2. LB broth
      3. DMSO
      4. MgCl2
      5. Glassware & Equipment (Falcon tubes; 500μl Eppendorf tubes, on ice; 200ml conical flask; 200μl pipetman or repeating pipettor; 5ml pipette)
      6. Cells
   2. Procedure
      1. TSS buffer (125 mL): dissolve 25 g PEG 8000 in final volume of mL 105 LB broth filter sterilize, add 12,5 mL DMSO and 7,5 mL 1M MgCl2
      2. 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.
      3. In the morning You should aim to dilute the overnight culture by at least 1/100.
      4. 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)
      5. 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).
      6. Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
      7. All subsequent steps should be carried out at 4°C and the cells should be kept on ice whenever possible.
      8. Centrifuge for 10 minutes at 3000 rpm and 4°C.
      9. Decant supernatant, remove leftover media by carefully pipetting
      10. Resuspend in TSS buffer (10% of original volume), vortex gently
      11. Add 100 μl aliquots to chilled Eppendorfs, flash freeze and store at – 80°C in 200 µl aliquots.
   3. Source
      1. Open wet ware
5. Making competent Lactococcus Lactis cells
   1. Materials
      1. M17 media
      2. Electroporation buffer
      3. Sucrose
      4. Glucose
   2. Procedure
      1. Grow cells overnight in 25ml of GM17 (M17 with 1% glucose)
      2. Add 1ml of overnight culture into 25ml SGM17 + 2.0% glycine
      3. SGM17 = 25ml M17 + 0.25g glucose + 5g Sucrose + 0.5g glycine)
      4. Grow for ~4 hours until OD600 ~ 0.7
      5. Chill culture on ice for 10 mins
      6. Centrifuge cells for 15 mins at 3000g
      7. Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol)
      8. Centrifuge cells for 15 mins at 3000g
      9. Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol)
      10. Centrifuge cells for 15 mins at 3000g
      11. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol)
      12. Separate into 100µl 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. Resuspend DNA
      2. Competent cells: 50 µl per transformation
      3. SOC Medium: Prepare fresh from fridge components
      4. LB+ antibiotic Plates: Two per transformation
      5. Eppendorf tubes- Floater
      6. Ice bucket
      7. 42°C water bath
      8. 37°C incubator: both shaker and stove
      9. Sterile spreader/glass beads
   2. Procedure (on ice)
      1. Thaw competent cells on ice. This may take 10-15min for a 260µl stock. 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 (labeled, prechilled, in floating rack), don’t forget control tubes
      3. Pipet 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. Heat shock tubes at 42°C for 30 seconds (precisely)
      6. Incubate on ice for 5 min
      7. Add 200 µl of SOC media to each transformation
      8. SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination
      9. Incubate at 37°C for 1 hours, shaker or rotor recommended
      10. Pipet 20µl & 200 µl transformation mixture onto petri plates and spread with sterilized spreader or glass beads
      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. http://parts.igem.org/Help:Protocols/Transformation
7. Electrotransformation Lactococcus Lactis
   1. Materials
      1. Electroporation Cuvettes
      2. Electroporator
      3. M17
   2. Procedure
      1. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes)
      2. Add 900µl ice cold M17 and keep on ice for 10 min
      3. To each ml of M17 add: 0.5M (.17g) Sucrose + 0.5% (15µl) Glucose + 20mM (10µl) MgCl2 + 0.2mM (10µl) CaCl2
      4. Subculture 100 µl into 900 µl prewarmed M17+ and incubate for 2 hours
      5. Plate with proper antibiotic
   3. 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
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
          NEB https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273
    3. 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	-

    4. Source
       1. https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
11. PCR cleanup, Gel extraction, minipreps was performed by using kits provided by Qiagen. Standard procedure was followed.
12. 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
13. Antibiotic
    1. Chloramphenicol
    2. Erythromycin
    3. Ampicillin