About ZR BAC DNA Miniprep Kit
The ZR BAC DNA Miniprep Kit is for the efficient isolation of BAC plasmid DNA or other large plasmids (e.g., PAC) from E. Coli using a procedure that is simple, rapid, user-friendly, and reliable. It features a modified alkaline lysis protocol with color-coded reagents that allow easy visualization and assessment of complete bacterial cell lysis and neutralization. The innovative Zymo-Spin™ IC-XL columns are optimized for high yield endotoxin-free plasmid DNA recovery. BAC DNA purified using the ZR BAC DNA Miniprep Kit is ideal for sequencing, PCR, endonuclease digestion, etc.
|Processing Time||15 min|
|DNA Purity||High-purity and endotoxin-free (<50 EU/μg) plasmid DNA in low salt buffer or water; typical A260/280 ≥ 1.8. Eluted DNA is suitable for sequencing, endonuclease digestion, PCR, and other reactions requiring highly purified DNA.|
|Plasmid DNA Size||Up to ~200 kb.|
|Plasmid DNA Yield||Up to 10 μg per preparation, depending on the plasmid copy number, initial volume of E. Coli culture processed,and culture growth conditions. The typical yield is 1 μg per 3 ml of culture.|
|Procedure||Can be conducted at room temperature, between 15-30°C.|
|Recovery Volume||≥ 10 µl|
HindIII and NotI digestion of BAC DNA. A BAC (~160 kb) from a RPCI-11 human BAC library (CHORI) was purified from DH10B cells (Invitrogen) using the ZR BAC DNA Miniprep Kit. Digestion with NotI removed the ~148 kb insert from the 11.6 kb pBACe3.6 cloning vector 1 (?). M: 1 kb DNA ladder (Zymo Research).
Spin down 0.5 – 5 ml of bacterial culture to a pellet at 5,000-6,000 x g for 5 minutes in an appropriate tube and centrifuge. Discard supernatant.
Add 200 µl of P1 Buffer (Red) to the tube and resuspend pellet completely (i.e., by vortexing or pipetting). Transfer to a 1.5 ml microcentrifuge tube.
Add 200 µl of P2 Buffer (Green)1 and mix by inverting the tube 4-6 times. Cells are completely lysed when the solution appears clear, purple, and viscous. Proceed to the next step within 3 minutes. Wait 1-3 minutes before proceeding to Step 4.
Add 400 µl of P3 Buffer (Yellow) and mix gently but thoroughly. Do not vortex vigorously. The sample will turn yellow when the neutralization is complete2. Wait 1-2 minutes before proceeding to Step 5.
Centrifuge sample(s) in a microcentrifuge for 3 minutes.
Place a Zymo-Spin™ IC-XL column into a Collection Tube and transfer the supernatant from Step 5 into the Zymo-Spin™ column. When transferring the supernatant avoid carrying any cellular debris to the column and be careful not to disturb the green pellet.
Centrifuge the Zymo-Spin™/Collection Tube assembly for 30 seconds.
Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin™ column to the Collection Tube3.
Add 200 µl of Endo-Wash Buffer to the column and centrifuge for 15 seconds.
Add 400 µl of Plasmid Wash Buffer4 to the column. Centrifuge for 30 seconds. Empty the Collection Tube and centrifuge for an additional minute.
Transfer the column into a clean 1.5 ml microcentrifuge tube and then add ≥10 µl of DNA Elution Buffer6 to the column. Centrifuge for 30 seconds to elute the DNA.