We performed heat shock transformation in chemically competent E. coli Top 10 cells during cloning stages, and again in BL 21 cells for protein expression. The latter included isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce protein expression (5). This reagent is omitted during transformation for cloning purposes. pET24d vector with Bst gene fragment insert, and BamHI and XhoI restriction enzymes were used. MiniPrep was completed as described in Thermo Fisher Plasmid DNA MiniPrep kit (6).

• Target DNA
• Chemically Competent Cells
• LB Stock Solution
• LB + kanamycin plate
• Thermo Fisher Plasmid DNA MiniPrep kit
• Bunsen Burner

1. Add 1 μL of plasmid into chemically competent cells. Gently pipette to mix. Incubate on ice for 30 min for colonies to grow.
2. Heat solution in 42°C water bath for 45 sec. Incubate on ice for 2 min.
3. Turn on Bunsen burner and leave on for the remainder of the procedure to keep the workspace sterile.
4. Swirl stock LB solution and add 1,000 μL of LB to a culture tube.
5. Add the entire volume of plasmid to the LB culture tube. Incubate at 37°C for 45 min.
6. Add 100 μL of solution to a LB + kanamycin plate. Spread solution evenly across the plate until most of the solution absorbs. Incubate at 37°C overnight.
7. Miniprep plasmid according to kit procedure.

BamHI and XhoI restriction enzymes were used for digestion of our modified Bst gene fragment into pET24d. Note: before digesting the vector and insert, restriction digest was performed on the vector with each enzyme individually and compared on an agarose gel against an uncut sample.

• 10X CutSmart Buffer
• Restriction Enzymes
• Target DNA
• SpectraMax® QuickDropTM Micro-Volume Spectrophotometer

1. Prepare reactions for gene insert and plasmid using the following volumes:

• 	Vector	Gene Insert
  10X CS buffer	5 μL	5 μL
  Restriction enzyme 1	2 μL	2 μL
  Restriction enzyme 2	2 μL	2 μL
  DNA	calculate (500 ng vector)	calculate (250 ng insert)
  Sterile water	variable	variable
  TOTAL VOLUME	50 μL	50 μL

2. Incubate samples at 37°C for 1 hour. Incubate samples at 80°C for 20 min.
3. PCR cleanup samples according to kit procedure to remove enzymes and unwanted buffer components.
4. Measure concentration of each sample by pipetting 2 µL of each into a sample port of the QuickDrop instrument.

• T4 Ligase
• T4 Ligase buffer
• Digested Plasmid
• Digested Insert

1. Prepare ligation reactions using the following volumes:

• 	Plasmid	Insert
  T4 ligase buffer	2 μL	2 μL
  T4 ligase	1 μL	1 μL
  Digested plasmid	calculate	calculate
  Digested insert	-	calculate
  Sterile water	variable	variable
  TOTAL VOLUME	20 μL	20 μL

2. Gently pipette up and down to mix. Incubate at room temperature for 30 min.

• LB Solution
• Crude Tagged Protein Induced with IPTG
• Crude Tagged Protein Not Induced with IPTG
• Lysis Buffer (50 mM Tris-HCl, 0.1 mM EGTA, 300 mM NaCl, 8% glycerol, 5 mM BME, PI tablet, 100 μL lysozyme)
• Wash Buffer (50 mM Tris-HCl, 0.1 mM EGTA, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 5 mM BME)
• Elution Buffer (50 mM Tris-HCl, 0.1 mM EGTA, 300 mM NaCl, 250 mM imidazole, 10% glycerol, 5 mM BME)

1. Prepare lysis, wash, and elution buffers.
2. Spin down LB solution and remove most of supernatant.
3. Resuspend the pellet and all 100 μL lysis buffer to induced sample and 50 μL lysis buffer to uninduced sample.
4. Incubate at room temperature for 30 min. sonicate for 15 sec, 3 times.
5. Wash with wash and elution buffers

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