Editing Plasmid MaxiPrep TMAE (section)

=== HIC Purification<br>  ===

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,<br> 

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For example:<br> 

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.<br> 

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Method Validation:<br> 

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.<br> 

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[[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 
| <br> 
| 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]] 

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

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

[[Category:Protocols]]