Editing Plasmid MaxiPrep Silica Oxide (section)

== Removal of RNA  ==

=== CaCl<sub>2</sub> Precipitation  ===

Precipitate RNA using CaCl2. Add 43 ul, 100 ul and 120 ul of 4 M CaCl2 to 300 ul filter solution from step 8, incubate at RT for 10 min, spin using microcentrifuge for 10 min.<br> 

Transfer supernatant to another tube. Add 300 ul isopropanol. incubate at RT for 15 min on bench, spin down pellet. Dissolve both Ca ppt and isopropanol ppt in 50 ul TE. Use 8 ul to run gel. 

[[Image:Ca ppt RNA-7 4-14.JPG]]<br> 

[[Image:Ca ppt plasmid 7 6 14.JPG]]SiO2_Plasmid_binding.JPG 

{| width="689" cellspacing="1" cellpadding="1" border="1"
|-
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br> 
| 7<br>
|-
| 
Without Ca ppt 

isopropanol ppt<br> 

| supenatant of 0.5 M CaCl2, ppt with isopropanol <br> 
| pellet of 0.5 M CaCl2 precipitation<br> 
| supenatant of 1.0 M CaCl2, ppt with isopropanol <br> 
| pellet of 1.0 M CaCl2 precipitation<br> 
| supenatant of 1.5 M CaCl2, ppt with isopropanol <br> 
| pellet of 1.5 M CaCl2 precipitation<br>
|-
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

<br> 

=== Acid Treatment of SiO2  ===

Add&nbsp; 0.1N HCl to silica oxide suspension, autoclave 30 min. Wash with water, use 50 ul to bind plasmid DNA from 300 ul of cleared lysate. 

7/7/2014<br> 

use acid washed SiO2 

[[Image:SiO2 Plasmid binding.JPG]]<br><br> 

1: +0.6 vol of isopropanol to bind to HCl washed SiO2<br> 

2: + 0.6 vol of isopropanol + 0.4% CTAB to bind to HCl washed SiO2<br> 

3:&nbsp; precipitate with isopropanol<br> 

7/8/14 

[[Image:SiO2 plasmid 7-8 14.JPG]][[Image:SiO2 plasmid 7-8 14 1.JPG]] 

<br> 

<br> 

{| width="379" cellspacing="1" cellpadding="1" border="1"
|-
| 1 
| No isopropanol + 0.1%CTAB, bind directly to SiO2
|-
| 2 
| 15% isopropanol + 0.1%CTAB, bind to SiO2
|-
| 3 
| 30% isopropanol + 0.1%CTAB, bind to SiO2
|-
| 4 
| 45% isopropanol + 0.1%CTAB, bind to SiO2
|-
| 5 
| 60% isopropanol + 0.1%CTAB, bind to SiO2
|-
| 6 
| 60% isopropanol + 0.1%CTAB + 50 mM NaH2PO4, bind to SiO2
|-
| 7 
| 60% isopropanol + 50 mM NaH2PO4, bind to SiO2
|-
| 8 
| 60% isopropanol, no SiO2
|}

===  ===

Disslove Isopropano precipitated DNA with PB, mix with 50 ul acid washed SiO2. 

[[Image:SiO2 plasmid 7-8 14 2.JPG<br>



{| width="283" cellspacing="1" cellpadding="1" border="1"
|-
| 2 
| use spin column to wash and elute
|-
| 1 
| use tube (no spin column)
|-
| 3 
| control, DNA before mixing with SiO2
|}

=== <br> ===

Large scale: from 250 ml culture<br>

[[Image:SiO2_plasmid_7_8_14_4.JPG]]<br>

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 1<br>
<br>

| large scale
|-
| 2
| mini scale<br>
|}



=== (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> Salt-Out <br>  ===

This is somebody's work, but I can not get it to work. After (NH4)2SO4 ppt, the supernatnat can not be processed with isopropano. Adding isopropanol will for large precipitate containing salt. Salt from 300 ul cleared lysate can be dissolved by 200 ul H2O<br> 

{| width="329" cellspacing="1" cellpadding="1" border="1"
|-
| Lane 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12
|-
| (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> Conc 
| 0 
| 0.4 
| 0.8 
| 1.2 
| 1.6 
| 2.0 
| 2.4 
| 2.8 
| 3.2 
| 3.6 
| 4
|}

<br> 

[[Image:Remove RNA ammoniumsulphate.jpg]] 

<br> 

=== 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