Editing Plasmid Miniprep 96 well

Buffers:

Resuspension Buffer (P1):<br> 50 mM Tris-HCl<br> 10 mM EDTA<br> pH 8.0<br> ** Note no RNase required for glass filter fiber purification***<br> 

Lysis Buffer (P2):<br> 0.2 M NaOH<br> 1% SDS (Sodium Dodecylsulfate)<br> 

Neutralization Buffer (P3):<br> 3 M KAc, pH 5.5<br> 

Binding Buffer (B1):<br> 7M Guanidine-HCL in Water<br> 

Wash Buffer (W1):<br> 70% Ethanol - 30% Water<br> 

Elution buffer (E1):<br> 1 X TE



Additional Materials:

Glass fiber filter: 800 µl filter plate from Polyfiltronics GF-B + 5.0 um Polypropylene for support



Lysate Clearing Procedure:

Start with 300 µl of saturated bacterial culture. Typically cells grown in TB for 24 hours to saturation in pressurized oxygen atmosphere. O.D. at 600nm ~18.<br> Harvest cells in 96 well flow-through microcentrifuge. Discard supernatant.<br> Resuspend cells in 100 µl of P1 by counter-rotation of centrifuge rotors.<br> Add 100 µl of P2 and mix gently.<br> Incubate for 5 min.<br> Add 100 µl of P3 and mix gently.<br> Pellet cell debris and K/SDS precipitate by spinning up rotors to 20,000g.<br> Collect the cleared lysate supernatant.

A) Ethanol Precipitation Procedure:

Add 300 µl 100% Ethanol to collected lysate and load into flow-through microcentrifuge.<br> Spin rotors up to 20,000g for 3 min. Discard supernatant.<br> Fill rotors with W1.<br> Spin rotors up to 20,000g for 1 min. to retain pellet. Discard supernatant.<br> Dry pellet with compressed air.<br> Add 60 µl of E1 and spin rotors up to moderate speed to dissolve pellet.<br> Collect purified template.

B) Glass Filter Procedure:

Add 300 µl of B1 to collected lysate and load into glass filter plate.<br> Incubate for 2 min.<br> Run lysate through filter using positive pressure above the plate.<br> Load filter with 600 µl of W1.<br> Run wash buffer through filter using positive pressure above plate.<br> Repeat wash steps twice more.<br> Flow compressed air through the filter in order to dry off any remaining ethanol.<br> Add 60 µl of E1.<br> Incubate for 2 min.<br> Elute from the filter under high pressure.<br><br>

[[Category:Protocols]]