Mike Barnkob:Protocols/Bacterial/Isolate DNA: Difference between revisions

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(New page: ← Front page ==Isolate DNA from bacteria== Diagnostic restriction digest to verify plasmid identity. ===Reagents=== * Restriction enzymes * DNA ladder ===Proto...)
 
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==Isolate DNA from bacteria==
==Isolate DNA from bacteria==


Diagnostic restriction digest to verify plasmid identity.
Protocol to isolate bacterial plasmids from overnight liquid cultures.


===Reagents===
===Reagents===


* Restriction enzymes
* QIAprep Spin Miniprep Kit.
* DNA ladder
* Autoclaved 1.5 mL Eppendorf tubes.


===Protocol===
===Protocol===


#  
''Follows QIAprep Spin Miniprep protocol. Presumes all reagens have been prepared.''<br />'''Note:''' All spins conducted at 13.000 RPM.
 
# Transfer bacterial culture into 2ml tubes.
# Spin for 3 min in mini-centrifuge.
# Remove supernatant and resuspend pellet in 250 μL Buffer P1 (kept at 4°C).
# Add 250 μL Buffer P2 and mix by inverting tubes 6-10 times, until solution turns blue.<br />'''Note:''' Finish this step in under 5 min.
# Add 350 μL Buffer N3 and mix by inverting tubes 6-10 times, until entire solution turns colorless.
# Spin for 10 min in mini-centrifuge.
# Transfer 750 μL of supernatant to spin column. Let sit for 60 sec minimum.
# Spin spin column for 45 seconds. Discard the flow-through by decanting.
# Add 500 μL Buffer PB and spin for 45 seconds. Discard the flow-through by decanting.
# Add 750 μL Buffer PE and spin for 45 seconds. Discard the flow-through by decanting.
# Transfer spin column to 1.5ml Eppendorf tube.
# Spin for 1 min to remove residual wash buffer.
# Transfer spin column to new 1.5ml Eppendorf tube.
# Add 50 μL Buffer EB (10 mM TrisCL) to the center of the spin column, very close to membrane. Let sit for 60 seconds minimum.
# Spin for 1 min. Flow-through is your DNA.
# Label tubes and store at -20°C
 
'''Quality control''':
# Add 1.2 μL of DNA to NanoDrop and record concentration, 260/280 and 260/230 ratios.


===References===
===References===


* http://www.addgene.org/recipient-instructions/diagnostic-digest/
* QIAprep Spin Miniprep Kit protocol.

Latest revision as of 02:48, 22 January 2015

← Front page

Isolate DNA from bacteria

Protocol to isolate bacterial plasmids from overnight liquid cultures.

Reagents

  • QIAprep Spin Miniprep Kit.
  • Autoclaved 1.5 mL Eppendorf tubes.

Protocol

Follows QIAprep Spin Miniprep protocol. Presumes all reagens have been prepared.
Note: All spins conducted at 13.000 RPM.

  1. Transfer bacterial culture into 2ml tubes.
  2. Spin for 3 min in mini-centrifuge.
  3. Remove supernatant and resuspend pellet in 250 μL Buffer P1 (kept at 4°C).
  4. Add 250 μL Buffer P2 and mix by inverting tubes 6-10 times, until solution turns blue.
    Note: Finish this step in under 5 min.
  5. Add 350 μL Buffer N3 and mix by inverting tubes 6-10 times, until entire solution turns colorless.
  6. Spin for 10 min in mini-centrifuge.
  7. Transfer 750 μL of supernatant to spin column. Let sit for 60 sec minimum.
  8. Spin spin column for 45 seconds. Discard the flow-through by decanting.
  9. Add 500 μL Buffer PB and spin for 45 seconds. Discard the flow-through by decanting.
  10. Add 750 μL Buffer PE and spin for 45 seconds. Discard the flow-through by decanting.
  11. Transfer spin column to 1.5ml Eppendorf tube.
  12. Spin for 1 min to remove residual wash buffer.
  13. Transfer spin column to new 1.5ml Eppendorf tube.
  14. Add 50 μL Buffer EB (10 mM TrisCL) to the center of the spin column, very close to membrane. Let sit for 60 seconds minimum.
  15. Spin for 1 min. Flow-through is your DNA.
  16. Label tubes and store at -20°C

Quality control:

  1. Add 1.2 μL of DNA to NanoDrop and record concentration, 260/280 and 260/230 ratios.

References

  • QIAprep Spin Miniprep Kit protocol.
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