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Hi Inna,
We usually have two lab sessions, each with 10 students. For
each session, we will need:
Inna's preparations:
- 6- VDX plates greased , place tape on the cover to be used
for a label (Inna)
- 180 -siliconized glass cover slips divided into six boxes
of 30 (Inna)
- 30 mL of 4M ammonium sulfate divided into six 5mL aliquots
(Inna)
- 6 mL of 1.0M Tris pH 7.0 and pH 8.0, each divided into
six 1 mL aliquots (Inna)
- 60 mL of distilled water divided into six 10 mL aliquots
(Inna)
- 6 boxes of yellow tips (Inna)
- 6 boxes of blue tips (Inna)
- 6 sets of pipetmen P-1000, P-100, P-20 (Inna)
- Large, Medium & Small gloves. (Inna)
- Coomassie stain and destain.(Inna)
- Parafilm and black sample-stamp-wells(Inna)
- Six 500 uL eppendorf tubes in a rack.(Inna)
- Tip disposal containers for each type of heavy atom. 2-Hg,
Sm, Au, Eu, 2-Pt, Gd, Ir, Er (Inna)
- Need two complete sets, one set for each half of the
lab bench.
- 6 homogeneous 12.5 gels (Inna)
- 6 sample loaders (6-well combs) (Inna)
- 12 native buffer strips (Inna)
Mike's preparations
- label 6 500uL tubes with 40mg/mL proteinase K (Mike)
- label 6 500uL tubes with 40mg/mL proteinase K + PMSF (Mike)
- label 6 500uL tubes with 2 mg/mL Proteinase K (Mike)
- make bench labels for groups A,B, and C. (Mike) /data1/users/sawaya/HTML/m230d/Crystallization/groupsigns.cdr
- 375 uL of 40 mg/mL proteinase K in 25 mM Tris pH7.0 divided
into six 62.5 uL aliquots (Mike)
- 375 uL of 40 mg/mL proteinase K in 25 mM Tris pH7.0 complexed
with 5 mM PMSF divided into six 62.5 uL aliquots (Mike)
- 120 uL of 4 mg/mL proteinase K divided into six 20uL aliquots
(if you use 2mg/mL you may not see the shifted band. (Mike)
- Make 100mM PMSF by weighing 0.0174 g PMSF, dissove in 1mL
isopropanol. (Mike)
- Weigh 20 mg (0.020g proteinase K) dissolve in 25uL 1M Tris
pH7.0 and 975uL water. BE SURE TO FILTER THE PROTEIN SOLUTION WITH 0.22um
filter. Otherwise you will get showers of crystals (Mike). Next
time, use greater than 40 mg/mL or else you will get porcupine crystals.
See notebook3 page 5.
- Take out 450uL proteinase K and add to 50uL PMSF. (Mike)
- Take out 16uL of proteinase K, add to 144uL water. Divide
into aliquots (Mike)
- 6 uL Aliquots of heavy atom solutions (Mike)
- Install the reverse polarity electrode in the PhastSystem
(Mike)
- reverse polarity electrode
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