Knight:PhoenIX Maxiprep Kit: Difference between revisions
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#Place PhoenIX™ Maxi column in the assembled column rack. | #Place PhoenIX™ Maxi column in the assembled column rack. | ||
#Place a beaker underneath to collect flow through. | #Place a beaker underneath to collect flow through. | ||
#Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to | #Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to drain by gravity flow. | ||
drain by gravity flow. | |||
#*''Note: It will take 15-20 minutes for the column to drain completely.'' | #*''Note: It will take 15-20 minutes for the column to drain completely.'' | ||
#Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C. | #Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C. | ||
#Remove all traces of liquid medium from the bacterial cell pellet by pouring. | #Remove all traces of liquid medium from the bacterial cell pellet by pouring. | ||
#*''Trace media can affect subsequent steps.'' | #*''Trace media can affect subsequent steps.'' | ||
#Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and | #Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended. | ||
vortex until completely resuspended. | |||
#Transfer resuspended cells to 50 mL conical tube. | #Transfer resuspended cells to 50 mL conical tube. | ||
#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube. | #Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube. | ||
Line 29: | Line 27: | ||
#Add 10 ml of neutralization buffer (green cap label). | #Add 10 ml of neutralization buffer (green cap label). | ||
#Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX. | #Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX. | ||
#*''Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of | #*''Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of the neutralization buffer before adding the buffer to the next tube.'' | ||
the neutralization buffer before adding the buffer to the next tube.'' | |||
#Centrifuge at 9,000 x g for 20 mins at room temperature. | #Centrifuge at 9,000 x g for 20 mins at room temperature. | ||
#''Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.'' | #*''Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.'' | ||
#Verify that the qquilibration buffer has been collected in the beaker. | #Verify that the qquilibration buffer has been collected in the beaker. | ||
#Discard the flow-through and replace the container. | #Discard the flow-through and replace the container. | ||
#Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column. | #Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column. | ||
#*''Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not | #*''Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not enter the column and cause clogging.'' | ||
enter the column and cause clogging.'' | |||
#Allow the lysate to drain by gravity flow (10-15 minutes). | #Allow the lysate to drain by gravity flow (10-15 minutes). | ||
#Discard the flowthrough and replace the empty container. | #Discard the flowthrough and replace the empty container. | ||
#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to | #Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes). | ||
drain by gravity flow ( | #Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes). | ||
#Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to | |||
drain by gravity flow ( | |||
#Discard the flow-through. | #Discard the flow-through. | ||
#Replace the waste collection container with a 50 mL conical tube. | #Replace the waste collection container with a 50 mL conical tube. | ||
Line 55: | Line 49: | ||
#Completely remove ALL of the supernatant from the pellet with a pipette. | #Completely remove ALL of the supernatant from the pellet with a pipette. | ||
#Air-dry the pellet for 10 minutes. | #Air-dry the pellet for 10 minutes. | ||
#''Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.'' | #*''Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.'' | ||
#Dissolve the plasmid DNA in 500 μl of water. | #Dissolve the plasmid DNA in 500 μl of water. | ||
#Move to smaller tube. | #Move to smaller tube. | ||
#Take a spectrophotometer reading to assess concentration. | #Take a spectrophotometer reading to assess concentration. | ||
==Notes== | |||
*These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g. | |||
*You're not supposed to let the column stand between steps. |
Revision as of 13:57, 10 December 2006
Materials
- PhoenIX™ Maxiprep Kit
- Isopropanol
- 70% Ethanol
- 5810R centrifuge
When using a new kit
- Assemble cardboard column rack.
- Note that the resuspension, lysis and neutralization buffer bottles are VERY hard to open.
Procedure
See official protocol. The steps below have been changed to accommodate our tubes and centrifuge.
- Place PhoenIX™ Maxi column in the assembled column rack.
- Place a beaker underneath to collect flow through.
- Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to drain by gravity flow.
- Note: It will take 15-20 minutes for the column to drain completely.
- Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.
- Remove all traces of liquid medium from the bacterial cell pellet by pouring.
- Trace media can affect subsequent steps.
- Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
- Transfer resuspended cells to 50 mL conical tube.
- Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
- Mix thoroughly by inverting until the lysate appears to be homogeneous (5-6 inversions). DO NOT VORTEX.
- Incubate 5 minutes at room temperature.
- Note: Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured.
- Add 10 ml of neutralization buffer (green cap label).
- Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.
- Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of the neutralization buffer before adding the buffer to the next tube.
- Centrifuge at 9,000 x g for 20 mins at room temperature.
- Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.
- Verify that the qquilibration buffer has been collected in the beaker.
- Discard the flow-through and replace the container.
- Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.
- Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not enter the column and cause clogging.
- Allow the lysate to drain by gravity flow (10-15 minutes).
- Discard the flowthrough and replace the empty container.
- Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
- Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
- Discard the flow-through.
- Replace the waste collection container with a 50 mL conical tube.
- Add 15 ml of elution buffer (pink cap label) to the top of the column.
- Allow the eluate to drain by gravity flow (5-10 minutes) into the centrifuge tube.
- Add 10.5 ml of room temperature isopropanol to the eluted plasmid DNA in the centrifuge tube.
- Mix and centrifuge at 9,000 x g for 40 minutes at 4°C.
- Pour out the supernatant taking care not to disturb the DNA pellet.
- Add 5 ml of room temperature 70% ethanol and wash the pellet.
- Centrifuge at 9,000 x g for 10 minutes at 4°C.
- Completely remove ALL of the supernatant from the pellet with a pipette.
- Air-dry the pellet for 10 minutes.
- Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.
- Dissolve the plasmid DNA in 500 μl of water.
- Move to smaller tube.
- Take a spectrophotometer reading to assess concentration.
Notes
- These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g.
- You're not supposed to let the column stand between steps.