CRI plasmid miniprep

General Info

This SOP is a how to use guide for purification of sequencing quality plasmid DNA.

Materials

• Avanti J20 rotor JS centrifuge
• Vortexer
• Eppendorf Centrifuge
• Speedy Vac
• Gilson pipettes – p200, p1000, p10 (and appropriate tips)
• Eppendorf tubes , 1.5ml.
• Racks to hold eppendorf tubes
• Orbital 37 degree C incubator shaker
• 10ml macartney bottles or 50ml corning tubes.
• Racks for holding the culture bottles in shaker.
• Supply of LB or 2x YT liquid culture (sterile)
• Appropriate Antibiotic.
• P1 , P2 , P3 (available from Qiagen : p1 cat: 19051, p2 cat: 19052 , p3 cat: 19053 each 500ml)
• RNase A cat: 19101 from Qiagen

Procedure

1. Inoculate a 10ml o/n culture of LB (2x YT is ok aswell) with a single distinct colony from a plate, add correct antibiotic for specific plasmid vector and grow o/n at 37˚C with shaking. Alternatively 10ul from freezing broth culture can be used.(Make sure labelling is clear and copied onto each culture vessel)
2. Centrifuge to pellet the cells for 20 mins at 2.5 k rpm, using Avanti J20 rotor JS centrifuge (JGL-SOP-058).
3. Pour off supernatant and re-suspend the cell pellet in 150ul of p1 (50mM Tris-HCL pH 8.0, 10mM EDTA and 100ug/ml RnaseA should be stored at 4 degrees centigrade).
4. Vortex to mix and transfer to 1.5ml Eppendorf tube – mix by vortexing there should be no cell clumps present
5. Add 150ul of p2 (200mMNaOH, 1% SDS if solution is cloudy heat to 37 degrees C until clear) mix by inverting DO NOT VORTEX lysis takes place the solution should go clear, leave for no longer than 5 mins at room temp.
6. Add 160ul of p3 (3.0M KoAc pH5.5) mix by inverting and leave on ice for 10 mins.
7. Centrifuge full speed 13,000 rpm in bench top eppendorf centrifuge for 30 minutes and then transfer the supernatant to fresh tube (Label this fresh tube clearly) this contains the plasmid DNA(Be careful not to disturb the pellet during the transfer, avoiding transfer of cell debris pellet).
8. Add 700ul of 100% isopropanol (propan-2-ol) mix by inverting.
9. After mixing centrifuge at max speed of 13k in Eppendorf centrifuge for 30mins.
10. Carefully take off the supernatant, DNA should be present as a pellet.
    1. Aspirate off all the liquid
    2. add 300ul of 70% ethanol and vortex then centrifuge for 10 mins at 13,000 rpm in Eppendorf centrifuge
    3. Decant off the 70 % Ethanol and discard. Allow the pellet to dry (2mins in the speedvac)
    4. Re-suspend the pellet in 33.25ul of water (nuclease free), pellet should be completely dissolved
11. Add 6.4ul of 5M NaCl and 40ul of PEG 8000 13% solution (autoclaved). Centrifuge down and mix by flicking with finger.
    1. Leave on ice for 20 minutes.
    2. Centrifuge at max speed 13,000 rpm in Eppendorf centrifuge (JGL-SOP-019), 30 minutes at 4 degrees C
    3. Aspirate off the supernatant (clear glassy DNA pellet should be present on the side of the eppendorf tube,take care not to disturb it.)
    4. Wash with 200ul of 70% Ethanol, mix by vortexing.
    5. Centrifuge at 13,000 rpm in Eppendorf centrifuge for 10mins
    6. Aspirate off the 70% ethanol and let the pellet dry (over night at room temperature is ok) or speed vac in 2-10 mins
12. After pellets are dry, re-suspend in 30ul of 10mM Tris pH 8.0 mix by vortexing and allow atleast 20minutes to dissolve.
13. Run out 1ul on a 1% Agarose gel along with control of known concentration from this estimate what amount to use in the sequencing reaction typically 1ul is needed. Stain the gel in 1l of 1xtbe containing 10ug/ml of Ethidium Bromide. Visualise the DNA under UV light.

RISK STATEMENT

Medium:

• Biohazard form the cell culture
• Use of Sodium Hydroxide, strong alkali causes burns.
• Ethidium Bromide is a carcinogen.
• Use of UV light, eye damage and harmful to skin.