Paul.xu: Created page with "Buffers: Resuspension Buffer (P1):
50 mM Tris-HCl
10 mM EDTA
pH 8.0
Note no RNase required for glass filter fiber purification*
Lysis Buffer (P2):
..."

2013-04-29T02:55:26Z

Created page with "Buffers: Resuspension Buffer (P1): 50 mM Tris-HCl 10 mM EDTA pH 8.0 ** Note no RNase required for glass filter fiber purification*** Lysis Buffer (P2): ..."

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Buffers:

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

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

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

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

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

Elution buffer (E1): 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. Harvest cells in 96 well flow-through microcentrifuge. Discard supernatant. Resuspend cells in 100 µl of P1 by counter-rotation of centrifuge rotors. Add 100 µl of P2 and mix gently. Incubate for 5 min. Add 100 µl of P3 and mix gently. Pellet cell debris and K/SDS precipitate by spinning up rotors to 20,000g. Collect the cleared lysate supernatant.

A) Ethanol Precipitation Procedure:

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

B) Glass Filter Procedure:

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

[[Category:Protocols]]

Plasmid Miniprep 96 well - Revision history

Paul.xu: Created page with "Buffers: Resuspension Buffer (P1): 50 mM Tris-HCl 10 mM EDTA pH 8.0 ** Note no RNase required for glass filter fiber purification*** Lysis Buffer (P2):..."

Paul.xu: Created page with "Buffers: Resuspension Buffer (P1):
50 mM Tris-HCl
10 mM EDTA
pH 8.0
Note no RNase required for glass filter fiber purification*
Lysis Buffer (P2):
..."