Lab Stocks

1M IPTG
238mg IPTG in 1mL dH20
Store at -20C

Lysozyme (use at 1mg/mL, dilute 100X)
100mg in 1mL dH20
Store at -20C

0.2M Chloromphenicol (use at 1mM, dilute 200x)
Dissolve 0.32g of chloramphenicol in 1mL 100% ethanol
Filter through 0.22um syringe filter
Store at -20C (stable at 4C for 2 years)
Inhibits hydrolysis of peptidyl-tRNA without disturbing much else
1


0.1M Phenylmethylsulfonyl fluoride (use at 0.1mM, dilute 1000x)
Dissolve 174mg PMSF into 10mL isopropanol
Store at -20C
Inhibits protease
2


0.5M CaCl2
Dilute 75mg into 1mL
Needed for DNase I (0.5mM)
3,4

500mM E Buffer (elution buffer for ribosomes, dilute 100x in R buffer)
107mg d-Desthobiotin (214g/mol)
1mL R buffer

500mL LB Broth
12.5 g LB
For plates, add 7.5 g agar
100mM d-Desthobiotin stock (use at 5mM, dilute 20x)
107mg d-Desthobiotin
5mL R buffer

25mM SM-(PEG)24
Mol weight = 1394.55
7mg SM(PEG)24
200ul DMSO

30mM HaloTag ligand
Mol weight = 328.9
5mg HaloTag ligand
506ul DMSO

30mM Cysteine
Mol weight = 121.2
3.64mg Cysteine
1mL PBS



Buffers

R buffer (500mL)
2.68g (25mM) Mg[OAc]2 (Mg++ keeps ribonucleoprotein together)
12.27g (250mM) K[OAc]
25mL (50mM) 1M Tris-HEPES, pH 7.0
475mL DEPC treated H20
R buffer (50mL)
5

50mL R buffer
10uL RNase Inhibitor
50ul TCEP (final: 0.5mM) (helps RNase Inhibitor work)
25uL EDTA (0.25mM) (needed to chelate divalent cations around peptidoglycans for lysozyme
to work)
50ul PMSF stock (0.1mM)
250ul chloramphenicol stock (1mM)
R buffer (1mL, before being diluted 4-6x)
1mL R buffer
6uL CaCl stock (3mM)
10ul DNase I
50ul Lysozyme stock (5mg/mL)
E buffer (3mL)
2.85mL R buffer
150ul 100mM d-Desthobiotin stock (final concentration: 5mM)
Sucrose cushion
20mL R buffer
5g sucrose (25% w/v, 22.2% w/w)
Protein extraction buffer
http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/enzymatic_lysis/
40ml for 1L cells
36ml H20
2ml 1M Tris-HCl (50mM Tris-HCL pH 7.6)
2ml Glycerol
350mg NaCl (150mM)
5ul 1M MgCl2
1ul 1mg/ml DNase
1mM PMSF
1mM TCEP
20mg Lysozyme (500ug/ml)
NPI-10 buffer (500ml)
http://rocfy.com/MichelleLab/penglab/Protocol/QIAGEN/Purification.pdf
50 mM NaH2PO4 3.45 g NaH2PO4H2O (MW 137.99)
300 mM NaCl 8.77 g NaCl (MW 58.44)
10 mM imidazole 0.34 g imidazole (MW 68.08)
Adjust pH to 8.0 using 5 NaOH granules

NPI-20 buffer (500ml)
http://rocfy.com/MichelleLab/penglab/Protocol/QIAGEN/Purification.pdf
50 mM NaH2PO4 3.45 g NaH2PO4H2O (MW 137.99)
300 mM NaCl 8.77 g NaCl (MW 58.44)
20 mM imidazole 0.68 g imidazole (MW 68.08)
Adjust pH to 8.0 using 4 NaOH granules
NPI-250 buffer (50ml)
http://rocfy.com/MichelleLab/penglab/Protocol/QIAGEN/Purification.pdf
50 mM NaH2PO4 0.35 g NaH2PO4H2O (MW 137.99)
300 mM NaCl 0.88 g NaCl (MW 58.44)
250 mM imidazole 0.85 g imidazole (MW 68.08)
Adjust pH to 8.0 using ?? NaOH granules

Protocols
Preparation of HaloTag ligand-linked mica (Taniguchi and Kawakami)
Cleave mica
Put in plastic sealed dish with 10uL of APTES and 3ul of diisopropylamine for 2 hours.
Store in room temperature in dessicator.
Add 5ul of 25mM SM(PEG)24 and 20ul of PBS to treated mica. Incubate for 30min.
Wash with water and isopropanol.
Add 1ul of 30mM HaloTag ligand and 20ul of PBS with 1mM TCEP and incubate 30min or
overnight.
Incubate with 50ul of cysteine solution (30mM in PBS) for at least 30 min.
Add 20ul of protein solution (1uM) to mica and incubate for 2hours at room temperature.

Isolation of RNCs from in vivo sample
Defrost RNC cells in room temperature water for 10min (thaw)
Aliquot into six 1.5mL tubes and add 166uL of R buffer to each tube. Let sit on ice for 10 min.
(lyse)
Freeze at -80C for 30min. (freeze)
Defrost at room temp water for 10min (thaw). Repeat 1x more freeze thaw cycle.
Spin at 13k @ 4C for 10 min. Collect supernatant in large pipette and pipette 500ul onto top of
25% sucrose cushion with R (carefully)
Spin at 80K for 64minutes.
Pipette out sucrose from top. Check to see if pellet exists! Pipette 200uL R buffer and aspirate
out to wash pellet. Then resuspend in 833uL R but swirly gently on ice by hand for a few
minutes.
Pour resuspended pellet into Strep-Tactin Sepharose column (exclusion limit > 6 x 10^6!!).
Make sure to have previously equilibrated column with R. Save flow through. Wash with 5mL
of R buffer. Save wash through.
Make 5ml of Elution buffer (50uL Buffer E 100x, 5ml R buffer). Let sit overnight.
The next morning, do another sucrose cushion with 1mL of sample on 2mL 25% sucrose at 80K
for 64 minutes.
Washing glass slides
Load glass slides into Teflon container (plastic calipers work best)
Sonicate on power 6 for 5 minutes in acetone.
Make Piranha solution, 5ml Hydrogen Peroxide, 15ml Sulfuric acid, under hood. Rinse out
acetone and pour in Piranha solution and turn heater to power 4-5 for one hour.
Pour Piranha into beaker under hood (be careful not to get acetone in the Piranha because it
forms acetone peroxide which is explosive). Fill with water and sonicate another 5 minutes on
power 6.
Take out glass slides and dry in air, put into vacuum.
Washing gold slides
Load glass slides into Teflon container (plastic calipers work best)
Sonicate on power 2 for 5 minutes in ethanol.
Sonicate on power 2 for 5 minutes in acetone.
Take out slides and dry in air, put into vacuum.

IPTG induction of protein
Day 1: Inoculate 25mL LB + 25ul 100mg/ml AMP with C41(D3)pLysS cells, grow at 37C overnight
Day 2: Inoculate 1L + 1ml 100mg/ml AMP with 12ml of overnight cells and grow at 37C for 3-4
hours. Then add IPTG to 0.2M (200ul of 1M IPTG) and shake overnight at room temperature.
Day 3: Spin down cells at 4C for 35min at 4100 rpm. Use column to purify
Preparation of carbon coated EM grids
Buy G400-Cu Electron Microscopy Sciences grids. They are 400 lines/inch square mesh grids.
Coat mica with carbon.
Put all the grids face up (dark side up). Add one drop of mixture of sticky tape soaked in
toluene to each grid. Let dry for one day.
Appendix
How long should I spin samples down?
5

Beckman TLA-100.3 rotor k-Factor @ 80000 RPM = 22.3
Viscosity S in 21% sucrose (w/w) @ 4C
6
= 3.3cP. Reduced S: 70S/3.3=21.2S
Time to spin (min.) = 60 * k-factor / reduced-S = 64 minutes
25% = 5g sucrose + 17 gram water = 21.7% surcorse
Spinning at 70K in 25% sucrose for 2 hours is the same as spinning in 90K in 35% sucrose for 20
minutes. David Reid has verified that 40S subunits are able to pellet in the shorter spin,
despite what equations say.




1. Tompkins, R.K., Scolnick, E.M. & Caskey, C.T. Peptide chain termination. VII. The ribosomal and
release factor requirements for peptide release. Proc Natl Acad Sci U S A 65, 702-8 (1970).
2. James, G.T. Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.
Analytical Biochemistry 86, 574-9 (1978).
3. Kunitz, M. Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric
method for the measurement of desoxyribonuclease activity. J Gen Physiol 33, 349-62 (1950).
4. Vanecko, S. & Laskowski, M., Sr. Studies of the specificity of deoxyribonuclease I. II. Hydrolysis of
oligonucleotides carrying a monoesterified phosphate on carbon 3'. J Biol Chem 236, 1135-40
(1961).
5. Moore. Ribosome Purification (OpenWetWare WIKI downloaded 03/22/2012). (2012).
6. Swindells, J.F., Snyder, C.F., Hardy, R.C. & Golden, P.E. Viscosities of Sucrose Solutions at Various
Temperatures: Tables of Recalculated Values. Supplement to National Bureau of Standards
Circular 440 (1958).