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PG for cryo-EM

Peptidoglycan purification for cryo-EM             Revised 20070724

The procedures detailed below were used to prepare peptidoglycan (sacculi) from Gram-negative bacteria like E. coli and C. crescentus, as reported in Gan et al. (2008).
Contains formatting errors for now.

Equipment
Hot stir plate
Volumetric flask, 100 ml
Beaker, 500 ml
Thermometer
SS34 rotor
2x Nalgene 40ml polycarbonate (transparent) bottles
4x Nalgene 40ml polypropylene (translucent) bottles


Stock solutions
4% & 8% w/v SDS (high quality, Fisher O2674-25)
1 mg/ml DNAse I
10 mg/ml Trypsin (T1426-250mg, TPCK treated, bovine pancreatic); stored @-20 ºC
1M MgSO4
10mM Na-Phosphate buffer, pH 7.8, 0.02% NaN3
2.5N NaOH (filtered)

4% SDS (RT)
10 g SDS (Fisher O2674-25)
250 mL ddH2O


10mg/ml trypsin, 1ml aliquots (-80ºC)
100 mg trypsin
10ml ddH2O


1 mg/ml DNAse I, 100ml aliquots (-20ºC)
100 mg DNAse I
100 mL 1mM HCl


2 mg/ml RNAse A, 200ml aliquots (-20ºC)
10 mg RNAse A
5 mL ddH2O


10mM Na-Phosphate buffer, pH 7.8, 0.02% NaN3 (500ml)
0.425 ml 1M NaH2PO4 • H2O
4.575 ml 1M Na2HPO4 • 7H2O
0.5 ml 20% NaN3
494.5 ml ddH2O
Adjust to pH 7.8 with NaOH or HCl


10mM Na-Phosphate buffer, 500ml (Adjust to final pH with NaOH or HCl)

1 L
500 ml

pH 7.0
pH 7.8
pH 7.0
pH 7.8


1M NaH2PO4 • H2O
3.9 ml
0.85 ml
1.95 ml
0.425 ml


1M Na2HPO4 • 7H2O
6.1 ml
9.15 ml
3.05 ml
4.575 ml


20% NaN3
1 ml
1 ml
0.5 ml
0.5 ml


ddH2O

989 ml

989 ml

494.5 ml

494.5 ml


Hot plate setting 1 = 54ºC; 2 = 65ºC; 3 = 79ºC



July 22, 2007



LB was made up with distilled water + LB Broth, Lennox (1426-2), 25g/L.



E. coli growth
Streak out frozen MG1655 (ATCC K-12 wild-type) E. coli onto LB plates.
Incubate @ 37ºC for ≥ 24 hours.
Transferred 2 colonies into 50ml of liquid starter culture (LB).
Grew until stationary phase.
Added 2 ml stationary culture to 500ml of fresh LB, No ampicillin!
Grew until OD600 =
Began crude purification.




*** Thawing on ice takes a LONG time, i.e. overnight ***


A. Freeze-thaw lysis: cells harvested @ OD600 = [July 24, 2007]

Vol.
Time


1. Cool 1L cells to 4ºC using ice (10 min).
10 m

2. Pellet cells @ 5,500RPM [4930xG / 5121.5xG] @ 4ºC, 15 min in GSA / GS3
15 m

3. Aspirate as much of the sup as possible without disturbing pellet

4. Re-suspend in 12ml ice-cold 100mM NaCl
15 m

5. Preheat water bath to 80ºC (5 min in microwave)
15 m




B. SDS lysis

Vol.
Time


1. Preheat 12 ml 8% SDS to 80ºC; prewarm SS34 to RT

2. Preheat 12 ml 4% SDS in a volumetric flask + stirbar to 80ºC water bath
12 ml

3. Add culture drop-wise to SDS solution (final [SDS] = 2%)
24 ml
15 m

4. Add 1/2 vol. 8% SDS 80ºC (final [SDS] = 4%)
36 ml
15 m

5. Continue heating ~ 60 min @ ≥ 80ºC
60 m

6. Split evenly into two Nalgene polypropylene SS34 bottles; top off with ddH2O.
60 ml

7. Centrifuge @ 43,223´G (19,000 RPM) @ 20ºC in SS34 rotor, 30 min.
60 ml
30 m

8. Resuspend + wash pellet 2x in 6 vol. ddH2O, pelleting in SS34 each time
60 ml
60 m

9. Resuspend in 9mL 10mM Phosphate buffer, pH 7.8 (can freeze @ -80ºC here)
9 ml


C. DNAse I + RNAse A + Trypsin digestion

Vol.
Time


1. Add MgSO4 to ~10 mM each (100 μl of 1 M stock) No CaCl2: It precipitates SDS!
9.1 ml

2. Add DNAse I to 10 μg/ml (100 μl of 1mg/ml stock)
9.2 ml

3. Incubate @ 30ºC, 30 min: DO NOT SHAKE; shaking inactivates DNaseI


30 m
4. Add RNAse A to 20 μg/ml (100 μl of 2mg/ml stock)
9.3 ml

5. Shake @ 30ºC, 225 RPM, 30m
30 m


6. Add trypsin to 1 mg/ml (1 ml of 10 mg/ml stock)
10.3ml

7. Shake @ 30ºC, 225 RPM, overnight
O/N


D. Second SDS treatment

Vol.
Time

1. Preheat 10 ml 4% SDS to 95ºC; pre-warm SS34 to RT

2. Dilute DNase/Trypsin-digested sacculi 3-fold with ddH2O
30 ml

3. Centrifuge @ 43,223´G (19,000 RPM) @ RT in SS34 rotor, 30 min.
30 m

4. Resuspend in 2 ml 4% SDS @ 95ºC
2 ml

5. Add suspension to 8 ml 4% SDS @ 95ºC, stir 30 min
10 ml
30 m

6. Add 20 ml ddH2O
30 ml

7. Centrifuge @ 43,223´G (19,000 RPM) @ RT in SS34 rotor, 30 min.
30 m

8. Resuspend in 30ml ddH2O and add 0.5ml 0.5M EDTA; pellet in SS34

9. Resuspend + wash pellet in 30 ml ddH2O; pellet in SS34 each time
60 m

10. Resuspend in 1 ml ddH2O, store @ -95ºC
1 ml


E. NaOH dissolution

Vol.
Time

1. Add 0.2 ml suspension (or resuspend pellet in) to 8 ml 2.5 N NaOH, preheated to 80ºC
10.5ml

2. Incubate 1 hr, w/ stirring
60 m

3. Add 20ml ddH2O; pellet @ 43,223´G (19,000 RPM) @ RT in SS34, 30 min.
30 m

4. Resuspend in 5ml 2.5N NaOH, add 25ml ddH2O

5. Pellet @ 43,223´G (19,000 RPM) @ RT in SS34, 30 min.
30 m

6. Resuspend in 30ml ddH2O; pellet @ 43,223´G (19,000 RPM) @ RT in SS34, 30 min.
30 m

7. Resuspend & pellet twice in 10mM NaxHyPO4 buffer (pH 7.0)
60 m­­­­­­­­­

8. Resuspend in a tiny quantity of phosphate buffer; store at 4ºC.