Editing Plant Genomic DNA Isolation Protocols (section)

== Silica&nbsp; powder (Celite 545 silica) method<br>  ==

'''Materials'''<br> 

Celite 545 silica powder (Celite 545-AW reagent grade) Sigma 20199-U<br> '''Buffers'''<br> 

<br> 

{| border="1" cellspacing="1" cellpadding="1" width="680"
|-
| DNA BINDING BUFFER<br> 
| 6M Guanidine thiocyanate <br>MW = 118.2 g/mol <br>70.92 g / 100mL (6M)<br> 
| &nbsp;!!! it takes several hours until dissolved (leave it approx. 4-5 hours)
|-
| WASH BUFFER<br> 
| 1mL of 5M NaCl + 99mL of 95% EtOH<br> 
| &nbsp;!!! PREPARE FRESH, because the salt<br>precipitates during storage<br>
|-
| LYSIS BUFFER (standard)<br> 
| 0.5% SDS (w/v) in 10x TE<br>0.5g SDS /100mL<br> 
| <br>
|-
| 5M NaCl stock solution<br> 
| MW=58.44g/mol <br>29.22g / 100mL<br> 
| Do not use if percipitate forms. Either heat to get fully back into solution or 5M NaCl stock solution<br>discard and make fresh<br>
|-
| 3M Sodium acetate (pH = 5.2)<br> 
| MW=82.03g/mol 
24.61g / 100mL<br> 

| Adjust pH value with glacial acetic acid<br>
|-
| DNA ELUTION BUFFER<br> 
| Tris-EDTA (TE) buffer, 10mM Tris-HCl, pH=8.0, 1 mM EDTA<br> 
| <br>
|-
| 10X&nbsp;TE<br> 
| Composition: 100mM Tris-HCl, pH=8.0 10 mM EDTA<br> 
| <br>
|}

<br> 

'''PREPARATION OF SILICA POWDER-DNA BINDING SOLUTION'''<br><br>Fill silica powder (Celite 545 silica) into 50 mL Falcon-tube (to about 2.5mL = approx. 800mg)<br>Add 30 mL dH2O<br>Shake vigorously (vortex and invert)<br>Let slurry settle for approx. 15 min<br>Remove (pipette off) the liquid<br>Repeat 2 times (a total of 3 washes)<br>After last washing step: resuspend the silica powder in about the same amout of water(up to about 5 mL)<br><br>STORE the silica solution at RT until further use (silica&nbsp;: H 2 O = 1&nbsp;: 1)<br>Before use:<br>suspend stored silica solution (silica&nbsp;: H 2 O = 1&nbsp;: 1) by vortexing<br>Transfer ~50 μL of silica solution to 2mL tubes (prepare 1 tube per sample)<br>!! try to keep the silica suspended during pipetting to ensure an equal distribution<br>Add 1mL H2O (a final wash step)<br>Mix by vortexing<br>Centrifuge: full speed (13,200) for 10-20 sec<br>Pipette off liquid<br>Add 700 μL DNA binding buffer (6M Guanidine thiocyanate)<br>Suspend the silica powder in DNA binding buffer<br>The silica binding solution is now ready for further use in the protocol (see Methods)<br><br> 

'''PREPARATIONS'''<br>&nbsp;Prepare 2 mL tubes (1 per sample): add 3 metal beads (tungsten carbide beads) per tube<br>&nbsp;Harvest leaf material (starting amount of material: about 100 mg fresh weight)<br> 

'''GRINDING'''<br>Use appropriate / available grinding protocol (Mortar &amp; pestle, Qiagen TissueLyser)<br>e.g. grinding using Qiagen TissueLyserII:<br>&nbsp;Freeze 2mL tubes containing leaf material and 3 metal beads in liquid nitrogen<br>&nbsp;Grind in TissueLyser by shaking (10 sec at 1/30 speed)<br>&nbsp;Re-freeze in liquid nitrogen (&gt;30 sec)<br>&nbsp;Grind again in TissueLyser by shaking (10 sec at 1/30 speed)<br>&nbsp;Re-freeze in liquid nitrogen (&gt;30 sec)<br>&nbsp;Keep in liquid nitrogen until lysis buffer is added or store at -80°C for later use<br> 

'''LYSIS'''<br>&nbsp;Add 800 μL Lysis buffer<br>&nbsp;Add 4 μL RNaseA (100 mg/ml)<br>&nbsp;Vortex (~2 min until the powder is dissolved in the buffer)<br>&nbsp;Incubate: 10min at room temperature<br>&nbsp;Add 200 μL 3M Sodium Acetate (pH = 5.2)<br>&nbsp;Mix by inversion of tubes<br>&nbsp;Incubate on ice for 5 min<br>&nbsp;Centrifuge 13,200 rpm / 5 min / RT (pellet the leaf material)<br> 

'''DNA BINDING'''<br>&nbsp;prepare 700 μL silica binding solution (see above)<br>&nbsp;transfer 800 μL of the supernatant to the tubes containing silica binding solution)<br>!! Do not transfer leaf material!<br>&nbsp;Completely resuspend the silica powder by vortexing and inversion of tubes (ca 20 sec)<br>&nbsp;incubate 15 min at RT (on a shaker at 400 rpm and/or invert tubes from time to time)<br>&nbsp;Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)<br>&nbsp;Remove the supernatant (with pipette)<br>WASHING (2 times)<br>&nbsp;Add 500 mL wash buffer (prepared fresh)<br>&nbsp;Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)<br>&nbsp;Centrifuge 13,200 rpm / 3 min / RT (pellet the silica)<br>&nbsp;Repeat the washing step (optional: a third washing step)<br>&nbsp;Remove the supernatant with pipette (as complete as possible)<br>&nbsp;optional: short spin and remove residual liquid with pipette<br>&nbsp;After last washing step: dry the silica in the hood up to 1 hour at RT (make sure there is no wash buffer left)<br><br> 

'''RESUSPENSION'''<br>&nbsp;Add 200μL TE buffer or 10mM Tris buffer<br>&nbsp;Completely resuspend the silica powder by vortexing and inversion of tubes (approx. 20 sec)<br>&nbsp;Incubate: 20 min / RT / with gentle agitation (on a shaker at 400 rpm and/or invert tubes from time to time)<br>&nbsp;Centrifuge (for tubes): 13,200 rpm / 5 min / RT (pellet the silica)<br>&nbsp;transfer 180 μL supernatant to new tube (avoid transferring silica powder!)<br>&nbsp;optional: if there is still silica powder in the preps – repeat the centrifugation<br>&nbsp;check for concentration and integrity of DNA<br>&nbsp;store the genomic DNA at -20°C for long-term storage or 4°C for short-term storage<br>