Editing Plasmid MaxiPrep Silica Oxide (section)

=== CTBA precipitate  ===

Cetyltrimethylammonium bromide (CTAB) is a nonionic detergent that can precipitate nucleic acids and acidic polysaccharides from '''low''' ionic strength solutions (CTAB will precipitate very little plasmid DNA from P1+P2+P3 solution). Meanwhile, proteins and neutral polysaccharides remain in solution under these conditions. In solutions of <u>high ionic strength, CTAB will not precipitate nucleic acids and forms complexes with proteins</u>. CTAB is therefore useful for purification of nucleic acid from organisms which produce large quantities of polysaccharides such as plants and certain Gram-negative bacteria, also remove endotoxin.<br> 

7/6/2014<br> 

[[Image:Ca ppt plasmid 2014 6 6.JPG]]<br> 

{| width="700" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Sample<br>
|-
| 1, 15<br> 
| 0.4% CTAB precipitate<br>
|-
| 2, 16<br> 
| isopropanol ppt<br>
|-
| 3,17<br> 
| 60% isopropanol ppt<br>
|-
| 4<br> 
| 0.25M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 5<br> 
| 0.25M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA<br>
|-
| 6<br> 
| 0.5M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 7<br> 
| 0.5M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA<br>
|-
| 8<br> 
| 0.75M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 9<br> 
| 0.75M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA<br>
|-
| 10<br> 
| 1M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 11<br> 
| 1.5M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 12<br> 
| 2M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt<br>
|-
| 13<br> 
| 1.5M (NH4)2SO4, 30 min, spin 10 min at 25 oC, supernatant, ppt with 0.4% CTAB <br>
|-
| 14<br> 
| 2M (NH4)2SO4, 30 min, spin 10 min at 25 oC,&nbsp; supernatant, ppt with 0.4% CTAB <br>
|}

<br> 

experiment 2 

<br> [[Image:CTAB ppt plasmid 7 6 14.JPG]] 

{| width="700" cellspacing="1" cellpadding="1" border="1"
|-
| Lane<br> 
| Sample<br>
|-
| 1<br> 
| Isopropanol precipitate<br>
|-
| 2<br> 
| 0.5 M CaCl2 ppt<br>
|-
| 3<br> 
| 0.5 M CaCl2+ 60% isopropanol ppt<br>
|-
| 4<br> 
| 0.5M CaCl2 ppt<br>
|-
| 5<br> 
| (0.4% CTAB + 0.5 M CaCl2) ppt<br>
|-
| 6<br> 
| Supernatant of #5 (0.4% CTAB + 0.5 M CaCl2), ppt with isopropanol <br>
|-
| 7<br> 
| 0.4% CTAB ppt<br>
|-
| 8<br> 
| supernatant of #7, ppt with isopropanol<br>
|-
| 9<br> 
| 0.8% CTAB ppt<br>
|-
| 10<br> 
| 
supernatant of #9, ppt with isopropanol 

|}

the disapearence of DNA from CTAB samples is probably due to washing with 70% ethanol after CTAB precipitation. When CTAB is present DNA could be soluble in 70% EtOH