Comparative Physiology @ Guelph
TnT Purification - 2008
- Rat cTnT in Pet24a(+) plasmid
- Plasmid is resistant to Kanamycin
- working concentration of Kanamycin is 60 ug/ml
- To start growth create starter culture: at 5 PM innoculate 2 x 50 ml volume of LB (with Kanamycin) with freshly transformed bacteria.
- grow overnight at 37 oC, 200 rpm
- Put the 6 X 1 litres of TY in shaker so they are ready next morning
- early next morning add 10 mls of LB starter culture to each litre of TY (with kanamycin)
- grow at 37 oC 250 rpm until OD600 = 0.6-0.8
- Innoculate each litre with IPTG working concentration = 0.4 mM
- up a 6 Liter pellet,
- collect pellet into single bottle
- Freeze overnight
- Bring volume up to 250ml with 50mM Tris pH 7.4, 0.1% TritonX100, 1mM PMSF
- Mix well, you may need to use a Polytron if it is very thick
- Transfer to a glass beaker and while stirring add drop by drop
- 25mg freshly made lysozyme (have it at 10mg/ml in water = 2.5ml solution)
- Keep stirring until it gets sticky, then continue stirring for 3-4 hours at room temp or overnight in the cold room at 4°
- Sonicate on #10 setting in multiple 2 minute bursts on ice until it gets less sticky
- Centrifuge at 14,000 for 20-30 minutes, save super and discard pellet
- Measure volume of super and add 16.4g/100ml Ammonium Sulfate (30%)
- Stir to dissolve and check pH, do not let pH drop below 7.0 (pH 7.5 is ideal)
- Centrifuge at 14,000 for 20 minutes and save super and discard pellet
- Measure volume of super and add 12.7g/100ml Ammonium Sulfate
- Centrifuge at 14,000 for 20 minutes, save pellet and discard super this time
- Dissolve pellet in 25-30ml DE buffer
- Dialyze this in 1 Liter DE buffer, changing 2-3 times
Kanamycin:
need enough antibiotic for 6200 ml of buffer
= 6000 TY for expression
– 100 LB for starter culture
– 100 LB for agar plates
working concentration of Kanamycin is 60 ug/ml
total: 0.372 g
disolve antibiotic (salt) in 6.2 ml water
filter sterilize through syringe filter.
Store frozen in tube wrapped in tinfoil.
add 1 ml/litre of media
IPTG
working concentration = 0.4 mM
MW = 238.3
need 0.95 g/litre of media
for 6 litres need 0.571 g
disolve in 6 ml of water and filter steralize
add 1 ml to each litre
DE Column Buffer
25 mM Tris
6 M urea
1 mM EDTA
15 mM BME
pH 8.0
Elute using 0-250 mM NaCl (in DE Buffer). Eluted at around 0.2 M NaCl
Dialyze peak fractions into CM buffer
CM Buffer
6M Urea
25mM MES
1mM EGTA
15mM BME
pH 6
Elute using 0-0.3 M NaCl (in CM Buffer). Eluted at around 0.2 M NaCl.