Plasmid Miniprep using CTAB

Plasmid DNA CTAB Miniprep from E. coli Preparation

Label 2 Eppendorf tubes per sample, turn heat block on to 100°C or prepare boiling water bath, prepare fresh lysozyme, put CTAB(5%) in warm water bathwith stirring to make sure crystals completely dissolved. This protocol works fine in the summer, then I discovered it was less good during the colder months –until I realizedthat the CTAB was dropping out of solution instead of precipitating the plasmid. The best way to avoid this is to carry out the procedure without refrigerationuntil you get rid of the CTAB in step 11. 
Procedure
1. Set up a 3-5mL overnight in a 20mL sterile culture tube with appropriate selection –remembering to inoculate with a single colony. 
2. Spin down cells (enough to give cell pellet, but not so much that it is difficult to resuspend) –remove supernatant(clear, yellow) 
3. Re-suspend each sample of cells in 0.4 mL STET (8% sucrose w/v, 0.1% v/v Triton X-100, 50mM EDTA, 50mM Tris pH 8.0) 
4. When all samples are re-suspended add 20ul 50 mg/mL lysozyme (store on ice until use) 
5. Invert until well mixed, and incubate at room temperature until 5 minutes after the addition of lysozyme, or until viscosity increasing markedly (no more than 10 minutes), pierce lids 
6. Boil samples on heat block for exactly 45 seconds (must be about 100°C), can use boiling water bath if large number of samples!) –I find a boiling water bath is better than a heating block (and quicker to heat up!). 
7. Spin down immediately 15 minutes at full speed (cell pellet contains chromosomal DNA), move supernatantto clean tube 
8. Add 20ul CTAB (5% w/v) to each sample, and mix by inversion

Final CTAB concentration 20*5/(440+20)=0.217%

if successful, a white precipitate will immediately form, and you should see this hanging in suspension in the sample. Incubate at 40°C for 5 minutes without shaking to maximize yield. If there is none visible in a particular sample, then no plasmid will be recovered. I think this improves yield because CTAB itself precipitates in the absence of DNA at lower temperatures (about 20°C) 
9. Spin for 3 minutes at full-speed 
10. Remove supernatant completely, but do not let samples dry out 
11. Resuspend each sample with CTAB-DNA with 300ul 1.2M NaCl (may require vortex) 
12. Add 780ul ‘absolute’ ethanol to each sample, mix by inversion until mixing is complete. 
13. Spin10 minutes at full-speed. 
14. Remove supernatantand wash pellet with 70% ethanol (breaking up pellet) 
15. Spin-down washed pellet 5 min.at full speed, and remove ethanol wash 16.Let remaining 70% ethanol evaporate (i.e.Air-dry), to dryness (may take an hour ormore on a cold day)

Isolation  730 mg of purified, supercoiled plasmid DNA. 

Cell lysis
600 mL of cell paste was thawed using warm tap water and resuspended in 5.4 L of STET buffer. 
The 10-fold dilution yielded a slurry with an optical density of 30 at 600 nm. 

Add 40 mcL of Ready-Lyse Lysozyme (Epicentre, 30 kU/mcL) to the cell slurry. 

Incubate the diluted cell slurry  at approximately 40° C. for 45 minutes while mixing

Heat lyse the cells  through an electro-polished, stainless steel coil immersed in a boiling water bath. 

The flow rate was adjusted so that the temperature of fluid exiting the heat exchanger is roughly 70° C. 

The lysis coil was then cleaned with pyrogen-free water, immersed in an ice water bath, and used to cool the hot lysate to 30° C. 

Lysate was cooled within 30 min of the completion of heat lysis. 

The total lysate volume was 5.6 L.

Clarification

Celpure P300 (Advanced Minerals) at a body feed concentration of 30 g/L was used to clarify the cooled lysate. 

Celpure P300 was mixed into the lysate, and the resulting slurry was divided into four portions. 

The amount of Celpure can be varied over a wide range depending upon the final scale of operation, the desired filter size and configuration and the designed production rate of the manufacturing facility. 

Similar considerations dictate the quantities of diatomaceous earth in the low cut and high cut filtration steps described herein. 

Each portion was filtered separately through a 25-micron stainless steel mesh contained within a 6-inch diameter filter housing. 

The filtrate was initially recirculated until its turbidity decreased to approximately 10 NTU. Following each filtration, pressurized air was used to displace interstitial fluid within the filter cake. 

The total volume of clarified lysate was 5.05 L. 

The total DNA concentration and purity of supercoiled plasmid DNA, as measured by an analytical HPLC assay, were 0.338 g/L and 83.6%, respectively.

CTAB Probe

A rapid CTAB probe was employed to determine the approximate low and high cut CTAB concentrations. Incremental amounts of 1.0% w/v CTAB in 40 mM NaCl were added to 500 mcL aliquots of clarified lysate in 1.5 mL glass vials. Vials were vortexed and visually inspected for the presence of DNA precipitates. 

Based on the probe results, low and high cut CTAB concentrations of 0.23 and 0.30% w/v, respectively, were assigned.

Low cut CTAB step

To 5.05 L of clarified lysate a 1.5 L solution of 1.0% w/v CTAB in 40 mM NaCl was added at room temperature over a 43 minute time period. 34.2 g of Celpure 300 was then added. The slurry was then filtered through a 25-micron stainless steel mesh contained within a 6-inch diameter filter housing. Filtrate was initially recirculated until its turbidity was constant. The filter cake was not washed but was dried using pressurized air. The final volume of product-containing filtrate was 6.44 L.

High cut CTAB step

29.3 g of Celpure P300 was added to the low cut filtrate. With agitation, 650 mL of 1.0% w/v CTAB in 40 mM NaCl was then added to the slurry over a time period of roughly 30 min. Analytical IPLC analyses revealed that all of the DNA had precipitated. The high cut slurry was then filtered through a 25-micron stainless steel mesh contained within a 6-inch diameter filter housing. The filtrate was recirculated until a constant turbidity was observed. The filter cake was washed with a 1 L solution of 0.3% w/v CTAB in 12 mM NaCl after the filtration of the high cut slurry was completed. Two 500 mL wash fractions were collected. The washed cake was then partially dried using pressurized air, collected from the filter housing, and weighed to determine the mass of residual liquid within the cake. The total mass of cake was 113 g. Celpure and precipitated DNA contributed approximately 29 and 2 g, respectively, indicating that there was roughly 82 mL of residual liquid in the washed cake. FIG. 2 shows a concentration profile during step precipitation with CTAB. These data show that plasmid DNA precipitates over a tight detergent increment. This CTAB-precipitation step is selective for removal of protein, RNA and endotoxin, which remain soluble.

Selective redissolution of washed, high cut cake—700 mL of sterile water was added to the washed, high cut cake, bringing the total volume of liquid to approximately 782 mL. Roughly 86 mL of 5 M NaCl was added to the slurry, yielding a NaCl concentration of approximately 0.5 M and redissolving the supercoiled plasmid DNA. Residual diatomaceous earth was filtered through a 25-micron stainless steel mesh contained within a 6-inch diameter filter housing to separate the Celpure from the redissolved supercoiled plasmid DNA. The filtrate was recirculated until a clear filtrate was obtained. The filter cake was washed with approximately 1 L of 0.5 M NaCl to recover interstitial, product-containing liquid. The 0.5 M NaCl wash was collected in four fractions. An analytical HPLC assay was used to assay the product filtrate and the four wash fractions for total DNA concentration and supercoiled plasmid DNA purity. The volume, total DNA concentration, and composition of supercoiled DNA of the product filtrate and 0.5 M NaCl washes were the following: (i) product filtrate: 800 mL, 1.933 g/L, 93.0%, (ii) wash fraction 1:200 mL, 0.203 g/L, 89.1 %, (iii) wash fraction 2:200 mL, 0.047 g/L, 70.3%, (iv) wash fraction 3:300 mL, 0.018 g/L, 61.4%, and (v) wash fraction 4:300 mL, 0.015 g/L, 63.3%. The wash fractions were not added to the product filtrate due to their low purity and concentration.