Digestion of Constructs

Papain Digestion Solutions

• 1. PBE buffer - Phosphate (100mM)(40%diNa;60%monoNa), EDTA (10mM), pH 6.5

Add 7.1g Na₂HPO₄, 1.86g Na₂EDTA to 500ml dH₂O

(Na₂HPO₄ 142 g/mol, Na₂EDTA.H₂O, 372 g/mol)

Adjust to pH 6.5 with conc. HCl

Filter sterilize

Store at 4°C

• 1. Papainase (type III, 19U/mg) (Worthington #P-3126)

Make fresh: papain (125g/ml), cysteine (10mM*), phos(100mM), EDTA (10mM), pH 6.3 *MW cysteine-HCl =175.6g/mol

Dissolve 0.035g cys in 20ml PBE

Filter Sterilize

Add 0.10ml sterile papain stock (25mg/ml)

(use TB syringe and needle; alcohol wipe stock enzyme)

Proteinase K Digestion Solutions

• 1. Stock solution TRIS, EDTA

To make one liter: 6.055g TRIS (50mM) in ~950 ml H₂O)

Adjust pH w/ 6N HCl to 7.6

Add 0.372 g EDTA x 2 H₂O (1mM)

Add H₂O up to 1000 ml

• 1. Solution A (working solution)

100ml from TRIS, EDTA stock

add 18.5 mg iodoacetamide (1mM)

add 1 mg pepstatin A (10g/ml)

heat to dissolve –takes a while

• 1. Solution B (working solution)

50ml from solution A

add 50 mg of proteinase K (1mg/ml)

incubate in 56°C water bath overnight (~16hr)

Digestion

• 1. Weigh wet constructs

  2. Freeze dry constructs

  3. Weigh dry constructs

  4. Add digestion solution to vials containing constructs

     1. 1 ml for 2mm thick sample

     2. 2 ml for 5mm thick sample

  5. Incubate in 60°C water bath for 10 to 16 hrs

  6. Vortex

  7. Refreeze until ready for assays