Triplex DNA can be used as substrates for the analysis of a variety of DNA motors, various triplex forming sequences have been identified and once formed can be exchanged into a desired buffer for analysis/assay.

Recipe:

Protocol:

To anneal, mix equimolar concentrations of linear (dsDNA duplex) and TFO in a 1X concentration of the above buffer.  Heat to 57˚C for 15 minutes and slowly cool to room temperature (preferably overnight).

Blue Native loading buffer can be used to load buffers on non-denaturing gels.  The 10 X Blue Native Loading dye give proteins and complexes a charge based upon their size and is compatible with a variety of native gel systems.

Recipe:

Protocol:

Samples are prepared as with other gel loading dyes (i.e. to a final conecentration of 1X) and native gels run as standard. Due to the nature of the loading dye, the gels can be visualised directly without post staining techniques, but are still suitable for Western blotting or 2D electrophoresis.

Medias required (for component recipes go here):

ZYP-0.8G (for starter cultures)

• Rich medium for growth with little or no induction
• Culture should go somewhat acid at saturation (slightly below pH 6)
• Collect cultures for freezer stocks well before saturation
• For all media, add 1 M MgSO₄ before adding 20xNPS to avoid precipitate
• Kanamycin is used at significantly higher concentrations (100 µg/ml) than is normally the case (25-40 µg/ml). Studier has found that in the T7 expression strains in these rich media, it does not provide adequate selection at the lower concentrations.
  

ZYP-5052 rich medium for auto-induction (bacterial growth and expression of proteins)

• For all media, add 1 M MgSO₄ before adding 20xNPS to avoid precipitate
• Kanamycin is used at significantly higher concentrations (100 µg/ml) than is normally the case (25-40 µg/ml). Studier found that in the T7 expression strains in these rich media, it does not provide adequate selection at the lower concentrations.
• Use 400 ml in a 2 liter baffled flask (if available).
  Adequate aeration is essential to the performance of this media. Don't use more than 20% of the nominal volume of the flask.
  Baffled flasks will give significantly better performance. You might obtain adequate results with non-baffled flasks, but I don't recommend it.

Autoinduction protocol

• Set up starter cultures of the bacterial colonies harbouring the plasmid to be used for overexpression in 2ml ZYP-0.8G (with an appropriate antibiotic) and grow over the day at 37°C until the cultures begin to look moderately cloudy (~6-8hours).
• Use 200µl of starter culture to inoculate 400ml of ZYP-5052 and grow cultures overnight at your chosen temperature (If growing at lower temperatures growth and expression can take significantly longer to reach endpoint).
• In the morning (after ~16hrs) recover bacteria by centrifuging at 12,000 rpm for 12 minutes and store cell pellets at -20°C until required (Average pellet =~3.5g per 400ml culture).

Autoinduction Stock Solutions (for Protocol go here):

ZY

• 10 g N-Z-amine AS (or any tryptic digest of casein, e.g. tryptone)
• 5 g yeast extract
• Make to 1 litre with dH₂0
  

20x NPS

• Add in sequence in beaker; stir until all dissolved.
• pH of 20-fold dilution in water should be ~6.75.
  

50x5052: (5052 = 0.5 % glycerol, 0.05% glucose, 0.2% α-lactose)

• Add in sequence in beaker, stir until all dissolved
• Lactose is slow to dissolve — may take two hours or more of stirring. Brief heating in a microwave can speed up dissolution of the lactose.
  

1 M MgSO₄

• 24.65 g MgSO₄•7H₂O
• Water to make 100 ml
  

40% glucose (w/v)

• Add glucose to stirring water in beaker; DO NOT ATTEMPT TO ADD WATER TO GLUCOSE!
• Stir until all dissolved — may take 45 minutes or more of stirring.
  

80% glycerol (v/v) (= 100% w/v)

• 100 g glycerol (weigh in beaker)
• 20 ml water
  

20% α-lactose (w/v)

• add lactose to stirring water in beaker
• stir until all dissolved -- may take 2 hours or more
  

1000x trace metals mixture (100 ml in ~50 mM HCl)

Prepare all metal stock solutions in ddH₂O, except for FeCl₃, which is dissolved in ~0.1M HCl, as noted in the table below. Combine the metal solutions as in the table below.

Autoclave the stock solutions of the individual metals, except 0.1 M FeCl₃ in 1/100 volume conc HCl. A brief precipitate may appeared upon addition of Na₂SeO₃, however it will redissolve rapidly. Store at room temperature

When making growth media, add the metals mix before NPS. If NPS is already present when 1000x metals mix is added, a precipitate forms which disperses but retains a light turbidity. If the metals are diluted to near their final concentration before NPS is added, the medium remains clear. The metals also precipitate and disperse or redissolve when added to ZY, a precipitate caused by yeast extract. Although apparently not a problem, the precipitate could be avoided by diluting the metals in the water before dissolving the yeast extract in making ZY.

Stock Solutions

M9 minimal salts solution (5X concentrate)

• To 800mL of distilled/deionized water add:
• 64g Na₂HPO₄-7H₂O
• 15g KH₂PO₄
• 2.5g NaCl
• 5.0g NH₄Cl
• Make to 1 litre with dH₂0
• Sterilze by autoclaving or filter sterilization if autoclave is not available.

1 M MgSO₄

• 24.65 g MgSO₄•7H₂O
• Water to make 100 ml
  

40% glucose (w/v)

• 40g Glucose
• Water to 100ml
• Add glucose to stirring water in beaker; DO NOT ATTEMPT TO ADD WATER TO GLUCOSE!
• Stir until all dissolved — may take 45 minutes or more of stirring.

1M CaCl₂

• 147.014g CaCl₂·2dH₂0
• Make to 1 litre with dH₂0

Method

To prepare 1L of media, add:

• 200mL 5X M9 salts solution
• to ~800mL of distilled water
• Add 15g of agar media if agar plates are to be poured.

After autoclaving, swirl to mix evenly and "temper" at room temperature (until you can place your hand on the flask for 2 second) then add:

• 2mL of 1M MgSO₄ solution
• 0.1mL of 1M CaCl₂ solution
• 20mL of 20% glucose

NOTE: The following media is used for growth of cultures for the overproduction of biotinylated fusion proteins using the AVBIOL cell strain, which is transformed with a plasmid encoding the BirA gene (for production of Biotin Ligase) available from Avidity.com (original protocol is available here).  In order to take advantage of this method of biotinylating proteins you must have an oligonucleotide sequence encoding the biotinylation recognition sequence upstream or downstream of your expressed gene.

Media:

TYH Media, 1 Liter

• 20 g tryptone
• 10 g yeast extract
• 11 g HEPES
• 5 g NaCl
• 1 g MgSO4
• pH 7.2-7.4 with KOH

Protocol:

1. Grow a 10 ml overnight culture from a single colony or glycerol stock in TYH media supplemented with 10 μg/ml chloramphenicol and the appropriate antibiotic (eg. ampicillin) needed to maintain the expression vector with shaking at 37˚C.
2. Place 5 ml of the overnight into 1 L of TYH media in a baffled Fernbach flask with 100 μg/ml ampicillin. Note: Chloramphenicol is not included.
3. Add 20 ml of a 20% sterile glucose solution (0.5% final conc.) and shake vigorously at 37˚C.
4. When the OD600 of the mixture reaches 0.7, remove 1.5 ml as a pre-induction sample.
5. Add 10 ml of 5 mM biotin solution (50 μm final). The biotin solution is made by adding 12 mg of d-biotin to 10 ml of warm (microwaved) 10 mM bicine buffer (pH 8.3) and filter-sterilizing the solution with a syringe and a 0.2 micron filter.
6. Add 15 ml of 100 mM IPTG (1.5 mM IPTG final) to induce for 3 hr.
7. Pellet cells in 4 x 250 ml centrifuge bottles at 5858 x g for 10 min.
8. Pour off media from cell pellets, re-suspend each pellet in 10 ml B-PER (Pierce Chemical Company, Pittsburgh, PA) (40 ml total volume).
9. Shake on a rotary shaker 10 min, RT.
10. Combine suspensions into one bottle and centrifuge at 16,270 x g for 15 min.
11. Save supernatant. Re-suspend pellet in 25 ml B-PER. Shake on a rotary shaker 10 min, RT.
12.  Centrifuge at 16,270 x g for 15 min.
13. Add supernatant to that previously saved. Discard pellet.

Pre-induction and induced samples of bacterial proteins can be analyzed by SDS-PAGE, western blotting with labeled StrepAvidin, or enzymatic means.