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Antibiotics

Prepare stock solutions of antibiotics for adding to media (1 µL per 1 mL)

Ampicillin 100 mg/mL in water
Chloramphenicol 50 mg/mL in ethanol
Kanamycin 30 mg/mL in water

Media Preparation

LB media

- 10 g/L Tryptone
- 5 g/L NaCl
- 5 g/L Yeast Extract
- 15 g/L Agar (solid media only)

One sleeve (20 plates) can be made with 600 mL of solid media (autoclave, cool, and add antibiotics before pouring)
One rack (72 16x100 mm tubes with 4 mL) can be made with 300 mL of liquid media (autoclave after pouring)

SOC media

- 20 g/L Tryptone
- 5 g/L Yeast Extract
- 0.5 g/L NaCl
- 950 mL/L ddH2O
pH to 7.0, autoclave, cool, and add the following
- 5 mL/L 2M MgCl2 (filter sterilize)
- 20 ml/L 20 mM Glucose Final Concentration* (Add 0.018 g/mL and filter sterilize)

TSS Method for Competent Cell Preparation

TSS Solution

Use the following recipe to make 100 mL:
- PEG 4000 15 g
- 1 M MgCl2-solution 5 mL
- LB liquid media add to 95 mL
- DMSO 5 mL (add after autoclaving)
Adjust pH to 6.5 prior to autoclaving.
After addition of DMSO aliquot TSS solution in 10 – 15 mL portions and store at –20 0C (TSS can get contaminated very quickly).

Competent Cell Preparation

Cultivate overnight E. coli culture* (*LB, add appropriate antibiotics if competent cells containing a plasmid for co-transformation are required) to inoculate main culture* 1:100 with overnight culture.
- Note: 50 mL culture will give 10 aliquots of competent cells, Use larger culture volumes (e.g. 100 mL) to prepare more aliquots.
Grow main culture at 37 0C and 260 rpm to ensure rapid growth to OD 0.4 – 0.6 (typically 2 – 3 hours, fast growing cells to OD 0.4 reach highest transformation efficiencies)
Centrifuge cells for 10 min at 4000 rpm (4 0C)
Carefully resuspend cell pellet in cold (4 0C) TSS solution (2 mL TSS for each 50 mL culture volume).
Incubate resuspended cells for 5 min on ice and aliquot 200 µL competent cells in 15 mL sterile tubes.
- Note: Handle cells carefully and keep them always on ice as they get very fragile during the TSS treatment.
Shock-freeze aliquoted cells in liquid nitrogen and store cells at –80 0C.

Restriction Digest

Prepare the following reaction mixture for a double digest:
- 3 µL Appropriate 10X Buffer (choose to maximize activity efficiencies)
- 1 µL Restriction Enzymes (two, 1 µL each)
- 10 µL Template DNA with compatible restriction sites
- 16 µL ddH2O

Allow reaction to incubate for >2 hours or overnight at 37 0C
Inactive restriction endonucleases by heating at 65 0C for 10 min
Check results on agarose gel
Isolate DNA from appropriate sized band with gel purification kit (Invitrogen or GE)

Ligation

Prepare the following reaction mixture:
- 2 µL Ligase Buffer
- 1 µL T4 Ligase
- 5 µL Plasmid (Cut with restriction enzymes)
- 12 µL Insert (Flanked with restriction sits compatible with plasmid and cut with them)
Allow reaction to incubate overnight at room temperature
Transform reaction mixture

Primer Design

Primers are the 5’ ends of the sequence to be amplified by PCR
Choose primers that have similar melting temperatures (Tm) that are between 50 0C and 65 0C
Choose primers that have low complementation with sequence of interest
Restriction sites are normally introduced to the 5’ end of primers to aid assembly into vectors (BglII and NotI for BioBrick vectors)
Include a G or C nucleotide at the 3’ end
Primer sequences are reported and ordered in 5’ to 3’ direction
Reconstitute primer in 10 µL for every nmol reported on tube (100 pmol/µL final concentration)

Polymerase Chain Reaction

Prepare reaction mixture in 200 µL PCR Tubes with following recipe:
- 1 µL Template DNA
- 1 µL Each primer (Forward and Reverse)
- 5 µL 10X Thermopol Buffer
- 1 µL 10 mM dNTPs
- 2.5 µL 10 mM MgSO4
- 0.5 µL DNA Polymerase (Taq or Vent)
- 38 µL of Water to bring final volume to 50 µL

Program themocycler with the following:
Initial Denaturation 5 min 95 0C
Repeat 25 times

**Denaturation 30 sec 95 0C **Annealing 30 sec >3 0C below lowest primer Tm **Extention 1 min per 1 Kb 72 0C **Final Extention 5 min 72 0C **Storage ∞ 4 0C

Check reaction with 1% agarose gel (0.01 g/mL) in TAE buffer
Use 2% agarose gel when checking fragments <500 bp
If necessary, purify DNA using agarose gel purification kit

Transformation

Thaw competent cells at room temperature on ice
Add 1 µL of plasmid DNA or ligation reaction mixture to cells
Incubate on ice for 20 min
Heat shock at 43 0C for 40 sec
Add 800 µL of SOC to cells
Incubate at 37 0C for 1 hour
Plate 50 µL of culture or for ligations, spin down, remove 900 µL media, resuspend cells, and plate on solid media with appropriate antibiotic
Incubate at 37 0C for >18 hours
Pick colonies and transfer to 4 mL cultures with appropriate antibiotic (add antibiotic to liquid LB before cells)
Incubate at 37 0C for >18 hours
Use Miniprep kit (made by Promega) to purify plasmid DNA from overnight culture

Sequencing

Sequencing is conducted by the University of Minnesota Biomedical Genomic Center
Make 1:100 dilution (1 pmol/µL) of stock primers for sequencing purposes
Prepare the following mixture in 0.5 mL microcentrifuge tube:
- 4 µL diluted primer
- X µL Template DNA in vector (100 ng per Kb template, X = 100 x n Kb/DNA concentration (ng/µL))
- Y µL Sterile water to reach final volume of 13 µL
Name sequencing reaction with 3 letter prefix plus next highest unused number (ex. GEM01)


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Silicatein Activity Assay

Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) in 1 mM HCl to prepare prehydrolyzed silica particles. Stir at room temperature for 15 minutes to completely hydrolyze TMOS to silica monomers.
Prepare two reactions:

Reaction/Reagent	Prehydrolyzed TMOS Solution	Silicatein
Assay	1 mL	200 ng
Negative Control	1 mL	0 ng

Incubate at NIST standard temperature and pressure (1 atm, 20 ºC) for 2 hours.
Centrifuge sample (3,000 x g for 5 minutes) to precipitate polymerized silica partciles.
Decant supernatant containing unaggregated silica particles.
Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.

a) Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.

Remove aliquots of 10 μl and add to solution of 15 mL water and 1.5 mL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
Add 8 mL of reducing agent Metol to solution. Solution should turn a bluish color.
Record absorbance at 810 nm and compare to standard curve.

@@ Line 134: / Line 134: @@
 *Name sequencing reaction with 3 letter prefix plus next highest unused number (ex. GEM01)
 {{Team:Minnesota/Bottom}}
+=Silicatein Activity Assay=
+# Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) in 1 mM HCl to prepare prehydrolyzed silica particles. Stir at room temperature for 15 minutes to completely hydrolyze TMOS to silica monomers.
+# Prepare two reactions:
+{| class="wikitable"
+|-
+! Reaction/Reagent
+! Prehydrolyzed TMOS Solution
+! Silicatein
+|-
+| Assay
+| 1 mL
+| 200 ng
+|-
+| Negative Control
+| 1 mL
+| 0 ng
+|}
+# Incubate at NIST standard temperature and pressure (1 atm, 20 ºC) for 2 hours.
+# Centrifuge sample (3,000 x g for 5 minutes) to precipitate polymerized silica partciles.
+# Decant supernatant containing unaggregated silica particles.
+# Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
+# Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.
+:: a) Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.
+# Remove aliquots of 10 μl and add to solution of 15 mL water and 1.5 mL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
+# Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
+# Add 8 mL of reducing agent Metol to solution. Solution should turn a bluish color.
+# Record absorbance at 810 nm and compare to standard curve.

Team:Minnesota/Protocols