Glossary of Protocols

Making Competent Cells:

1. Plate DH5 alpha seed stock and grow overnight at 37C
2. Isolate colony from plate into LB broth tube and grow overnight
3. Inoculate 12.5 mL of SOB media with 150 uL of fresh overnight culture
4. Incubate SOB culture at 37C in shaking incubator for 2 hrs
5. Aliquot into 12 eppi tubes (1 mL per tube)
6. Put on ice for 10 minutes
7. Spin down for 10 minutes at 4000 rpm
8. Poor off all excess liquid (carefully pipette it out if needed)
9. Re-suspend pellet in 333 uL of TB per tube
10. Incubate on ice for 10 minutes
11. Spin down for 10 minutes at 4000 rpm
12. Poor off all excess liquid
13. Re-suspend pellet in 83 uL of TB per tube
14. A 5.83 uL of DMSO to each tube
15. Incubate on ice for 10 minutes

Notes:

• Prepare empty tubes by freezing at -20C before use
• Keep cells on ice as much as possible
• Divide into the required aliquots of 50 or 25 uL
• Store finished cells in -80C freezer

Gel Electrophoresis

1. Measure 0.5 g agarose powder and add it to a flask
2. Add 50 mL of TAE buffer to flask
3. Microwave flask or incubate in hot water bath until agarose solution has become clear
4. Let the solution cool to about 50-55C occasionally swirling to mix
5. Add 2-5 uL of Ethidium Bromide to agarose solution and swirl to mix
6. Secure the rails on either side of the gel tray in the up position and carefully pour agarose solution into casting tray.
7. Place combs in casting tray
8. Allow solution to cool and solidify
9. Loosen and lower rails on either side of the casting tray
10. Gently place tray in gel box.
11. Add TAE buffer until it is just over the surface of the gel
12. Carefully pull out the comb

Notes:

• To prepare samples, add 1 uL of Loading dye per 5 uL of sample
• Add 10-20 uL of sample to a given well
• Gel box is turned to 135 V and run until the darkest band reaches the center of the gel.

LB Agar plates

1. Add the following to an empty 1000 mL flask
2. 7 g of tryptone
3. 3.5 g yeast extract
4. 3.5 g NaCl
5. 10.5 g agar
6. Add Distilled H2O to final volume of 700 mL
7. Autoclave solution for 30-45 minutes on liquid cycle
8. Allow flask to cool in water bath
9. Add antibiotics when flask roughly 50-55C
10. Mix well by swirling
11. Label plates to be poured with media, antibiotic, and date poured
12. Pour plates, filling each with media until ~1/2-2/3 full flaming the mouth of the flask between each plate
13. Allow plates to cool at room temperature overnight
14. Put plates back into sleave, seal the sleave, and label it
15. Store plates in refrigerator

LB broth tubes

1. Add the following to a 1000 mL flask
2. 10 g tryptone
3. 5 g yeast extract
4. 5 g NaCl
5. Add distilled H2O to final volume of 1000 mL
6. Adjust to final pH of 7.0 with NaOH
7. Distribute into tubes (5 mL per tube) or bottles
8. Autoclave for 30-45 minutes on liquid cycle

SOB Media

1. Add the following to a 1000 mL flask or beaker
• 20 g tryptone
• 5 g yeast extract
• 0.6 g NaCl
• 0.2 g KCl
2. Add distilled H2O to final volume of 1000 mL
3. Autoclave for 35-40 minutes on liquid cycle
4. Add the following sterile reagents
   1. 10 mL of 1M MgSO4
   2. 10 mL of 1M MgCl2

Notes:

• It is recommended that SOB be made in smaller batches to prevent contamination of a large amount of it

SOC Media

1. To 1000 mL of SOB add the following:
2. 20 mL of 1M sterile glucose

Notes:

• See notes on SOB media

Reinforced Clostridial Media To a 1000 mL flask add the following:

1. 10 g beef extract
2. 10 g peptone
3. 5 g NaCl
4. 5 g dextrose
5. 3 g yeast extract
6. 3 g sodium acetate
7. 1 g soluble starch
8. 0.5 g L-Cysteine HCl
9. 0.5 g agar
10. Adjust pH to 6.8+/-.2 at 25C

Addition of resazurin is optional.

This media was prepared anaerobically with nitrogen gas in an anaerobic hood.



Antibiotics

1000x ampicillin

• 10mg/mL in distilled water

1000x chloramphenicol

• 34mg/mL in 100% ethanol

1000x X-gal

• 20mg/mL in DMSO

Antibiotic Spread Plates

1. Dilute the antibiotic of interest (25 uL of antibiotic in 75 uL of appropriate solvent)
2. Pipette 100 uL of the solution onto the each plate and spread with beads
3. The plates must be allowed to dry and the antibiotic soak in (this usually takes at least an hour)

Restriction Digest

--Protocol for double digestions with 1-3 total reations
• Recipe (for 25 uL reaction)

• 1000 ng DNA
• 2.5 uL Buffer (10x)
• 1 uL Enzyme 1
• 1 uL Enzyme 2
• X uL water (to total volume of 25 uL)

1. Place into thermocycler
2. Run program "digest" under the file "iGEM"

--Protocol for double digestions with Reactions(R)>=4 total reactions
• Cocktail Mix Recipe (for 20 uL reaction)

• 13 uL ddH2O per reaction --------> (13*R) uL ddH2O
• 2 uL Buffer (10x) per reaction -----> (2.0*R) uL Buffer (10x)
• 0.2 uL Enzyme 1 per reaction -----> (0.2*R) uL Enzyme 1
• 0.2 uL Enzyme 2 per reaction -----> (0.2*R) uL Enzyme 2

1. Add 15 uL Cocktail Mix to labeled 0.2 mL PCR tube
2. Add 5 uL DNA
3. Place into thermocycler
4. Run program "digest" under the file "iGEM"

Ligation

1. Add 11 µL of MilliQ H20
2. Add 2 µL from each sample you will be ligating (destination plasmid, and part at roughly 10 ng/µL)
3. Add 2 µL of 10x T4 DNA ligase reaction buffer
4. Add 1 µL of T4 DNA ligase
5. Mix well, and spin down
6. Incubate for 30 min at 16°C and 20 min at 80°C to heat kill
7. Use 2 µL of ligation to transform into competent cells

Chemical Transformation

The following protocol uses chemically competent cells made by the Missouri Miners iGEM team
1. Thaw 30-50 uL of chemically competent cells in micro-centrifuge tube on ice for 5 min
2. Add 1-2 uL of DNA to competent cell tube
3. Keep cells on ice for 30 min
4. Heat shock cells at 42C for 60sec
5. Place competent cell tubes on ice for 5 min
6. Add 250 uL SOC to competent cell tube
7. Tape tubes to the bottom of a 37C shaking incubator for 1-2hrs
8. Spread 20 and 200 uL volumes of "transformed" cells onto appropriately labeled plates with correct antibiotic and media
9. Place inoculated plates into 37C incubator for 12-16hrs

Plasmid MiniPrep

The following protocol uses reagants from the IBM plasmid mini-prep kit
1. Spin down LB broth cultures 1.5 mL at a time until most or all of the tube is gone.
2. Each LB tube will require at least one eppi tube
3. For each repetition of the previous step poor off the supernatant
4. Re-suspend the pellet in 200 uL of PD1 buffer
5. Add 200 uL of PD2 buffer and mix by inverting 10 times
6. Wait 2 minutes
7. Add 300 mL of PD3 buffer. Mix by inverting 10 times
8. Centrifuge for 3 minutes at 16xg
9. Place PD column into a 2 mL collection tube
10. Add supernatant from step 7 to the PD column and centrifuge at 16xg for 30 seconds
11. Discard flow-through and place the PD column back in the 2 mL collection tube
12. Add 400 uL of W1 buffer to PD column
13. Centrifuge at 16xg for 30 seconds
14. Discard flow-through and place PD column back in collection tube
15. Add 600 uL of Wash buffer to PD column
16. Centrifuge at 16xg for 30 seconds
17. Discard flow through and place PD column back in collection tube
18. Centrifuge again to dry at 16xg for 3 minutes
19. Transfer PD column to 1.5 mL eppi tube
20. Add 50 uL of MilliQ H2O for elution to the center of the PD column
21. Let stand for 2 minutes
22. Centrifuge at 16xg for 2 minutes
23. Discard PD column, close and label eppi tube.
24. Nano drop to determine concentration

Genomic MiniPrep The following are the protocols provided with the invitrogen PureLink Genomic DNA Mini Kit

Gram Positive Bacterial Lysate Protocol

1. Set two water baths or heat blocks at 55C and 37C respectively
2. Prepare Lysozyme Digestion buffer (see next recipe) to ~200 uL Lysozyme Digestion Buffer/sample, add fresh Lysozyme to obtain a final lysozyme concentration of 20mg/mL
3. Harvest up to 2x10⁹ Gram positive cells by centrifugation
4. Resuspend pellet in 180 uL of Lysozyme Digestion Buffer containing Lysozyme from step 2. Mix well by brief vortexing
5. Incubate at 37C for 30 minutes
6. Add 20 uL Proteinase K. Mix well by vortexing
7. Add 200 uL PureLink Genomic Lysis/Binding buffer and mix well by vortexing to yield a homogenous solution
8. Incubate at 55C for 30 minutes
9. Add 200 uL 96-100% ethanol to the lysate. Mix well by vortexing to yield a homogenous solution.
10. Proceed immediately to the purification protocol (after lysozyme digestion buffer preparation protocol)

Lysozyme Digestion Buffer Recipe

1. 25 mM Tris-HCl
2. ph 8.0
3. 2.5 mM EDTA
4. 1% Triton X-100

Purification Protocol
This purification procedure is designed for purifying genomic DNA using a spin column-based centrifugation procedure in a total time of 10-15 minutes

1. Remove a PureLink spin column in a collection tube from the package
2. Add the lysate (~640 uL) prepared with PureLink Genomic Lysis/Binding Buffer and ethanol to the spin column
3. Centrifuge the column at 10,000xg for 1 minute at room temperature
4. Discard the collection tube and place the spin column into a clean PureLink collection tube supplied with the kit
5. Add 500 uL Wash buffer 1 prepared with ethanol to the column
6. Centrifuge column at 10,000xg at room temperature for 1 minute
7. Discard the collection tube and place the spin column into a clean PureLink collection tube
8. Add 500 uL Wash Buffer 2 prepared with ethanol to the column
9. Centrifuge the column at maximum speed for 3 minutes at room temperature. Discard collection tube
10. Place the spin column in a sterile 1.5 mL microcentrifuge tube
11. Add 25-200 uL of PureLink Genomic Elution Buffer to the column. Choose the suitable elution volume for your needs.
12. Incubate at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute at room temperature
13. To recover more DNA repeat the elution step
14. Centrifuge the the column at maximum speed for 1.5 minutes at room temperature.
15. Remove and Discard the column, store purified DNA at 4C(short term) or -20C(long term)

PCR The following protocol requires bulls eye Taq Polymerase Master Mix. For each pcr reaction add to a single pcr tube the following reagents:

1. 12.5 uL Taq Master Mix (final concentration: 1x)
2. Forward Primer (final concentration: 0.1-1.0 uM)
3. Reverse Primer (final concentration: 0.1-1.0 uM)
4. Distilled or MilliQ water (to total final reaction volume of 25 uL)
5. Template DNA (4 uL of 1:50 diluted LB culture)
6. Total Volume of 25 uL Once the previous reagents have been mixed do the following:
7. Mix reaction gently by pipetting the solution up and down a few times
8. Run the PCR reaction using the following thermocycler program
   • 95C for 5 minutes
   • 95C for 30 seconds
   • 68C for 30 seconds
   • 72C for 45 seconds
   • 72C for 5 minutes
9. Steps b, c, and d should be run 20-30 times

Gel Extraction
This is the protocol provided with the IBI Gel/PCR DNA Fragment Extraction Kit

1. Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (TAE buffer is recommended go gel formation)
2. Transfer up to 300 mg of the gel slice to a 1.5 mL microcentrifuge tube
3. Add 500 uL of DG buffer to the sample and mix by vortex
4. Incubate at 55-60C for 10-15 minutes or until the gel slice has been completely dissolved. During incubation, invert the tube every 2-3 minutes
5. Cool the dissolved sample mixture to room temperature
6. Place the DF Column in a 2 mL collection tube
7. Transfer 800 uL of the sample mixture from the previous step to the DF column
8. Centrifuge at full speed for 30 seconds
9. Discard the flow through and place the DF column back in the 2 mL collection tube (if the sample is more than 800 uL, repeat the DNA binding Step)
10. Add 400 uL of W1 buffer into the DF column
11. Microcentrifuge for 30 seconds and then discard the flow-through
12. Place the DF column back in the 2 mL collection tube
13. Add 600 uL of Wash buffer (ethanol added)into the DF column and let stand for 1 minute
14. Microcentrifuge for 30 seconds and then discard the flow-through
15. Place the DF column back into the collection tube
16. Microcentrifuge again for 3 minutes to dry the column matrix
17. Transfer the dried DF column to a new 1.5 mL microcentrifuge tube
18. Add 15-50 uL of MilliQ H2O into the center of the column matrix
19. Let stand for 2 minutes or until the MilliQ is completely absorbed by the matrix
20. Centrifuge for 2 minutes at full speed to elute the purified DNA

TOPO TA cloning
The invitrogen TOPO TA cloning Kit was used for the following protocol. reactions are set up as described by the following:

1. Fresh PCR product: 0.5-4 uL
2. Salt Solution: 1 uL
3. MilliQ H2O: add to total volume of 5 uL
4. TOPO vector: 1 uL (final volume of 6 uL)
5. Mix reaction gently and incubate for 5-30 minutes at room temperature
6. Proceed to transformation and plating on X-gal spread plates or store overnight at 20C