Paul.xu at 21:46, 21 November 2012

2012-11-21T21:46:04Z

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== Fractogel EMD TMAE(M)  HiCap ==

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37oC.

2. Spin down cells, 

3. Resuspend cells with cold 10 ml to 20 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1 (with RNase A). Usually 15 ml P1 is good enough. Make sure all cells are suspended, no visible aggregate---This is very critical. You can use a clean spoon to disrupt the pellet. 

4. Lyse cells with 10 ml to 20 ml of P2/per gram of pellet. Usually 15 ml P2 is good enough. Swirl the centrifuge tubes gently until cells are completely lysed. Do not shake. 

5. Neutralize with cold 10 ml to 20 ml of P3/per gram of pellet. Usually 15 ml P3 is good enough. 

Make sure you mix the solution gently in the begining and then more vigorously, until all gooey lysate becomes loose  fluffy white material (lysate becomes not viscous at all). 

Alternatively 1M potassium acetate or 7M ammonium acetate without acetic acid can be used. Some people used this P3: (1.85M KOAc, 1.16N NH4OAc, and 1.15M NaCl pH 5.5) 

6. Incubate on ice (or store in the cold room) for 4 hours to o/n to precipitate RNA. This step is optional if P1 with RNAse is used, however, if P1 without RNase is to be used, this step is mandatory. In addition, it is not necessary to adjust the pH of P3 to 5.5. After neutralization the pH should be around 8. 

7. Spin at 6000 rpm for 10 min, then transfer nuetralized sol to 50 ml tubes, Spin at 12000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using a coffee filter. 

8. Add 1/20 volume (1.5 ml for 30 ml supernatant) of 20% Triton X-114 (in 80% ethanol), incubate on ice for 30 minutes, mix occasionally. 

1.2% Triton X-114+ 0.6% [w/v] SDS===> not cloudy at RT 

Other detergents: Nonidet-P40 (Igepal CA 630), Triton X-100, Tween 20 

9. Equilibrated a 7 ml TMAE column with 3 CV of '''Buffer EQ '''or Buffer A '''('''20 mM TRIS-HCl, pH 6.0, 1 mM EDTA, 600 mM NaCl, 10% (v/v) ethanol) 

10. Dilute DNA solution with 1 vol of water. Using a peristaltic pump, load DNA solution to  the column. flow rate should be less then 2 ml/min. Peristaltic pump: setting is 10 

11.  Wash  with 2 CV of '''Buffer A ''' (20 mM Tris-HCl, pH 6.0,  '''600''' mM NaCl, '''10% EtOH, 0.5% Triton X-114''' , 1 mM EDTA.

Buffer A: 

20 mM Tris-HCl: dilute Tris-HCl from 1 M Tris hydrochloric acid stock (no need to adjust pH).

600 mM NaCl: from 5 M Filtered NaCl

0.5% Triton X-114:  from 20% Triton X-114 in ethanol.

1 mM EDTA: from 0.5 M EDTA

 

This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260. 

This wash will elute endotoxin and residual RNA. 

12. Wash with 10 CV of  ml of '''Buffer B'''  (20 mM TRIS-HCl, pH 6.0, 1 mM EDTA, '''600 '''mM NaCl, 10% (v/v) ethanol or 10% isopropanol).  

This wash is to wash out Triton X-114 and residual RNA, nicked and lineared/relaxed circular plasmid. 

13. Elute plasmid DNA slowly (0.5  ml/min) using 3 ~4 CV of '''Buffer D''' ('''1.3 M''' NaCl, 20 mM Tris-HCl, pH '''10''', 1 mM EDTA), Discard the first 3 ml eluate, (usually it does not contain any DNA). Soak column in this buffer for 3 min after the first CV, then slowly (0.5 ml/min) elute DNA using peristaltic pump. Stop the pump every CV (7 ml) and soak the column for 5 min before next elution. Collect all eluate in a 50 ml tube. 

Alternative elution buffer: 50 mM TRIS, pH'''8.5, 1.5 M '''NaCl, '''15% (v/v) ethanol''', 1 mM EDTA 

'''Buffer D'''

1.3 M NaCl

20 mM Tris Base, no need to adjust pH.

1 mM EDTA

15.  Clean  column with 1 CV of 1 M NaOH, then 3 CV of 2.5 M NaCl, or once with 2 CV of CIP solution (1M NaCl+1.5 M NaCl). Wash out NaCl with 3 CV of water, Flush column with 2 CV 20% EtOH. Store the column in 20% EtOH at 4 oC. 

 16. Measure DNA concentration using nanodrop.

17. Precipitate DNA using 0.7 vol  of isopropanol, incubation on ice for 15 to 30 min.

18. Spin down DNA using high speed centrifuge 12000 rpm for 15 min.

Inside a biosafety hood:

19. Wash 2X with 2 ml 70% ethanol (by spray ~2 to 4 ml of 70% ethanol). Decant enthanol.

20. Dissolve DNA in 5 X 200 ul TE

21. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis. 

 

=== HIC Purification ===

If further purification using HIC resin is to be performed, 1.9M ammonium sulfate should be used to elute plasmid. (Step 14). If 1.3 M NaCl is used to elute plasmid, eluate must be adjusted to 1.3 M final concentration of ammonium sulfate. The eluate is then loaded to Octyl Sepahrose 4 FF column. At ~1.6M ammonium sulfate, plasmid flows through and residual RNA, endotoxins, and E. coli DNA bind. Alternatively, one can bind the TMAE pool in the presence of ~ 2.0M ammonium sulfate, which allows relaxed and/or nicked circular plasmid to flow through, while the supercoiled plasmid binds; the supercoiled can then be eluted with ~1.6M ammonium sulfate. Other HIC resin may also be used, such as Phenyl Sepharose, Pharmacia Butyl Sepharose, 

 

For example: 

The Tosoh phenyl 650-S resin (10 mL) was packed into a XK26 column. The column was equilibrated with approximately 50 mL of 1.75M (NH4)2SO4 at 2 mL/minute. Then 35 mL of the DEAE elution pool containing the plasmid was diluted with an equal volume of 3.5M (NH4)2SO4 which results in a final concentration of 1.75M (NH4)2SO4 . The entire 70 mL of plasmid DNA in 1.75M (NH4)2SO4 was pumped through the HIC column . The flow through peak contains desired plasmid. 

 

Method Validation: 

1 L culture of Clone 1382. 15 ml P1, 15 ml P2, 15 ml P3. Spin 6000 rpm for 5 min, then 12000 rpm for 10 min. Filter through coffee paper. Add 2 ml 20% TX-114, incubate on ice for 30 min. Load to 7 ml TMAE(m) HighCap column XK16. Wash with 15 ml Buffer A, 15 ml Buffer B, 50 ml Buffer C. ELute with Buffer D at 1 ml/min. 

 

[[Image:TMAE Plasmid 2 11 19 2012.jpg]]

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Fraction
| 1-4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
|-
| OD260
| N/A
| 420.3
| 421.6
| 190.6
| 104.5
| 75.4
| 66.2
| 57.5
| 43.1
| 38.8
|-
| 260/280
| 
| 1.81
| 1.82
| 1.84
| 1.81
| 1.80
| 1.78
| 1.78
| 1.73
| 1.70
|}

[[Image:TMAE Plasmid 11 19 2012.jpg]]

 

2012/11/21. 1382 Maxiprep by Yingying. Eluted fractions do not contain RNA, see below

[[Image:1382 2012 12 21.jpg]]

[[Category:Protocols]]

Plasmid MaxiPrep TMAE - Revision history

Paul.xu at 21:46, 21 November 2012