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Revision as of 22:44, 22 September 2011

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)