Griffitts:Miniprep: Difference between revisions

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* Qiaprep Spin Kit (27106)
* Qiaprep Spin Kit (27106)
* [[Griffitts:Common_buffers#TE_buffer|TE buffer]]
* [[Griffitts:Common_buffers#TE_buffer|TE buffer]]
* 1.5 mL microcentrifuge tubes (1 per culture)


==Procedure==
==Procedure==
#Grow DH5alpha with appropriate plasmid in liquid LB (4 ml) for 12-18 hours.
* Grow DH5α with appropriate plasmid in 4 mL liquid LB+[[Griffitts:Stock_solutions#Antibiotics|antibiotic]] for 12–18 hours
#Spin down 1.3 ml of culture, dump and tap out supernatant, and repeat.
* Label your spin columns and microcentrifuge tubes
#Resuspend pellet in 250 ul P1 (from fridge).
* Centrifuge 1.6 mL of each culture in a microcentrifuge tube at full speed for 30 seconds
#Add 250 ul P2, immediately invert 3 times, let stand for 2 min.
* Dump and tap out supernatant
#Add 350 ul N3, immediately invert 8 times.
* Resuspend pellet in 250 μL P1
#Spin 6 min.
** The P1 is kept in the fridge
#Carefully move supernatant (~820 ul) to spin column.
** Make sure to use P1 that has RNAse added
#Spin 30 s.
* Add 250 μL P2
#Remove flowthrough and add 650 ul of wash buffer PE to column.
* Immediately invert 3 times
#Spin 30 s.
* Let stand for 2 minutes
#Remove flowthrough.
* Add 350 μL N3
#Spin 60 s.
* Immediately invert 8 times
#Move column to 1.5 ml tube, add 50-100 ul TE directly to disk, let stand 1-2 min.
* Centrifuge for 6 minutes at full speed
#Spin 60 s to elute DNA.
* Carefully move the supernatant (~820 μL) to a spin column
* Centrifuge for 30 seconds at full speed
* Remove flowthrough
* Add 700 μL of wash buffer PE to column
* Centrifuge for 30 seconds at full speed
* Remove flowthrough
* Centrifuge for another 60 seconds at full speed
* Move column to a labeled 1.5 mL microcentrifuge tube
* Add 50–100 μL TE directly to disk
** Don't touch the membrane with your tip
* Let stand 1–2 minutes
* Centrifuge for 60 seconds at full speed to elute DNA


''Adapted from Qiagen handbook''
''Adapted from Qiagen handbook''

Latest revision as of 09:56, 16 May 2011


Standard Qiagen Miniprep

Materials

  • Qiaprep Spin Kit (27106)
  • TE buffer
  • 1.5 mL microcentrifuge tubes (1 per culture)

Procedure

  • Grow DH5α with appropriate plasmid in 4 mL liquid LB+antibiotic for 12–18 hours
  • Label your spin columns and microcentrifuge tubes
  • Centrifuge 1.6 mL of each culture in a microcentrifuge tube at full speed for 30 seconds
  • Dump and tap out supernatant
  • Resuspend pellet in 250 μL P1
    • The P1 is kept in the fridge
    • Make sure to use P1 that has RNAse added
  • Add 250 μL P2
  • Immediately invert 3 times
  • Let stand for 2 minutes
  • Add 350 μL N3
  • Immediately invert 8 times
  • Centrifuge for 6 minutes at full speed
  • Carefully move the supernatant (~820 μL) to a spin column
  • Centrifuge for 30 seconds at full speed
  • Remove flowthrough
  • Add 700 μL of wash buffer PE to column
  • Centrifuge for 30 seconds at full speed
  • Remove flowthrough
  • Centrifuge for another 60 seconds at full speed
  • Move column to a labeled 1.5 mL microcentrifuge tube
  • Add 50–100 μL TE directly to disk
    • Don't touch the membrane with your tip
  • Let stand 1–2 minutes
  • Centrifuge for 60 seconds at full speed to elute DNA

Adapted from Qiagen handbook

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