Protocols/Create

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==Overview==
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==Miniprep==
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Replace this sentence with a brief description of the protocol and its goal.
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This get you the DNA
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==Materials==
 
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For a 50 μL PCR reaction:
 
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* 35 μL H<sub>2</sub>O
 
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* 5 μL 10X PCR buffer
 
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* 5 μL 2mM dNTPs (each)
 
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* 1.5 μL 50mM MgCl<sub>2</sub>
 
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* 1 μL 50μM sense primer
 
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* 1 μL 50μM antisense primer
 
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* 1 μL 5nM DNA template
 
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* 0.5 μL TAQ DNA polyermerase
 
==Procedure==
==Procedure==
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# In a PCR tube, mix the components on ice in the order they are listed above.
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Collect a turbid sample of 10ml overnight growth
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# Perform thermocycling program
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# Centrifuge universal bottle for 10min 2600rpm
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## 95 °C 5 min
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# Discard supernatant
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## 95 °C 30 s
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# Resuspend cell pellets with 250μL of P1 Buffer by pipetting
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## T<sub>H</sub> 30 s
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# Transfer to sterile labelled eppendorf tube
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## 72 °C 1 min for each 1 kb PCR product
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# Add 250μL of P2 Lysing Buffer
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## Repeat steps 2-4 a total of 12-36 times (24 is standard).
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# Wait for 3-5 minutes (e.g. 3.5min)
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## 72 °C 5 min
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# Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
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## 12 °C hold
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# Centrifuge sample at 13K rpm for 10 minutes
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# Add supernatent to a nickle filter (ensure not solids)
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# Centrifuge for 1 minute (13K rpm) and discard flowthrough
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# Add 500μL PB to wash filter
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# Centrifuge for 1 minute (13K rpm) and discard flowthrough
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# Add 750μL PE (with Ethanol) for final wash
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# Centrifuge for 1 minute (13K rpm) and discard flowthrough
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# Centrifuge for 1 additional minute (13K rpm) and discard flowthrough
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# Transfer filter to sterile labelled eppendorf tube
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# Add 30μL Nuclease free water to centre of column
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# Wait for 1 minute
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# Centrifuge for 1 minute (13K rpm) to elute
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# Store sample on ice or in -20°C
==Notes==
==Notes==

Revision as of 14:33, 12 July 2012

Contents

Creating a New Protocol

  1. In the search box on the lefthand side navigation bar, type Protocol name. If it is a protocol specific to your lab, type LabName:Protocol name (be sure and replace LabName with your PI's last name like Knight or Smolke
  2. You should see a page come up with a message saying "There is no page titled "Protocol name". You can create this page." If you don't see this message then that means a page with that name already exists. You'll need to choose a different protocol name. Image:SearchforProtocol.tiff
  3. Click on the create this page link.
  4. View and copy the source from the protocols template into your new page and begin editing. Make sure you are not editing this page.
  5. Check your work by clicking the ShowPreviewButton.jpg button.
  6. Save the changes by clicking SavePageButton.jpg ... and you're off.


Miniprep

This get you the DNA


Procedure

Collect a turbid sample of 10ml overnight growth

  1. Centrifuge universal bottle for 10min 2600rpm
  2. Discard supernatant
  3. Resuspend cell pellets with 250μL of P1 Buffer by pipetting
  4. Transfer to sterile labelled eppendorf tube
  5. Add 250μL of P2 Lysing Buffer
  6. Wait for 3-5 minutes (e.g. 3.5min)
  7. Add 350μL of N3 Neutralisation Buffer - title well to ensure mixing (10-12 times)
  8. Centrifuge sample at 13K rpm for 10 minutes
  9. Add supernatent to a nickle filter (ensure not solids)
  10. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  11. Add 500μL PB to wash filter
  12. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  13. Add 750μL PE (with Ethanol) for final wash
  14. Centrifuge for 1 minute (13K rpm) and discard flowthrough
  15. Centrifuge for 1 additional minute (13K rpm) and discard flowthrough
  16. Transfer filter to sterile labelled eppendorf tube
  17. Add 30μL Nuclease free water to centre of column
  18. Wait for 1 minute
  19. Centrifuge for 1 minute (13K rpm) to elute
  20. Store sample on ice or in -20°C

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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