Team:Newcastle/Project/Labwork/PhilsProtocols

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(→Professor Phil Aldridge's Lab Protocols)
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# Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
# Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
# store at –80˚C until used up
# store at –80˚C until used up
 +
 +
 +
== Restriction Digests ==
 +
<b>In general</B>
 +
 +
* Solutions required:
 +
** ddH2O (sterile and filtered)
 +
** Restriction enzyme and buffer
 +
**Your DNA
 +
 +
 +
<b>Simple Test Digests</b>
 +
 +
* For simple test digests the final volume should be 20 ul.
 +
* If you are testing vector DNA, quantify using a UV spec and dilute an amount down (usually 200 ul) to 0.05 ug/ul and use this as your working solution.
 +
<Br>
 +
* All digests must contain the following:
 +
** Sterile H2O giving a final volume of 20 ul (usually 7.5 ul)
 +
** 2 ul 10x restriction buffer
 +
** 10 ul of your DNA
 +
** 0.5 ul Restriction Enzyme
 +
<Br>
 +
* All these should be added in the above order to prevent contamination.
 +
* Incubate at the appropriate temperature for 1 hour then run entire sample on a 0.8% agarose gel.
 +
<Br>
 +
<b>Cloning Experiments</b>
 +
* For digests during a cloning experiment all final volumes should be 50 ul.
 +
** Sterile H2O giving a final volume of 50 ul
 +
** 5 ul 10x restriction buffer
 +
** your DNA
 +
** 1 to 2 ul Restriction Enzyme
 +
<Br>
 +
* For your vector DNA digest 20 ul of your working 0.05 ug/ul solution.
 +
* If your insert is a PCR reaction digest the lot after cleaning it up or for a subcloning digest 1-2 ug of plasmid DNA.
 +
* Incubate all reactions at the appropriate temperature for 3 hours.
 +
* For inserts (whether PCR products or fragments) run entire reaction on a gel in 2 lanes and extract DNA using Sigma Gel Extraction Kit.
 +
* For vectors use a standard ethanol precipitation to get rid of buffer and resuspend pellet in 20 ul ddH20.
 +
<Br>
 +
* Use DNA immediately for a Ligation (different protocol sheet).
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Revision as of 23:48, 7 July 2009

Professor Phil Aldridge's Lab Protocols

Transforming DNA "Phil Style"

  1. Switch on heat block in flow to “LOW”.
  2. Go get cells out of -80˚C and leave on ice for 30 minutes.
  3. Check to see if heat block is at approx 42-45˚C.
  4. Add DNA 1-20 ul to cells after vortexing them.
  5. Leave on ice for EXACTLY 30 mins.
  6. Place tubes in heat block for EXACTLY 50 secs.
  7. Transfer back to ice for 2 mins.
  8. After 2 mins add 0.9 ml LB and incubate for 45-60 mins. At 37˚C.
  9. Plate out 200 ul and 200ul of a 1:10 dilution and start praying.


Preparation of cells

  1. Dilute a ON culture 1:200 into at least 200 ml LB
  2. Grow to an OD600 between 0.1 and 0.2 (usually 2-3hrs)
  3. Spin down cells
  4. Resuspend in 40ml ice cold 0.1 M CaCl2 and leave on ice for 30 min.
  5. Spin down cells and resuspend in 1 ml 0.1M CaCl2
  6. Transfer cells to an 1.7ml tube and add 105 ul glycerol and make sure you get a homogeneous solution
  7. Aliquot in 100 ul volumes into clean 1.7 ml tubes and shock freeze in liquid nitrogen
  8. store at –80˚C until used up


Restriction Digests

In general

  • Solutions required:
    • ddH2O (sterile and filtered)
    • Restriction enzyme and buffer
    • Your DNA


Simple Test Digests

  • For simple test digests the final volume should be 20 ul.
  • If you are testing vector DNA, quantify using a UV spec and dilute an amount down (usually 200 ul) to 0.05 ug/ul and use this as your working solution.


  • All digests must contain the following:
    • Sterile H2O giving a final volume of 20 ul (usually 7.5 ul)
    • 2 ul 10x restriction buffer
    • 10 ul of your DNA
    • 0.5 ul Restriction Enzyme


  • All these should be added in the above order to prevent contamination.
  • Incubate at the appropriate temperature for 1 hour then run entire sample on a 0.8% agarose gel.


Cloning Experiments

  • For digests during a cloning experiment all final volumes should be 50 ul.
    • Sterile H2O giving a final volume of 50 ul
    • 5 ul 10x restriction buffer
    • your DNA
    • 1 to 2 ul Restriction Enzyme


  • For your vector DNA digest 20 ul of your working 0.05 ug/ul solution.
  • If your insert is a PCR reaction digest the lot after cleaning it up or for a subcloning digest 1-2 ug of plasmid DNA.
  • Incubate all reactions at the appropriate temperature for 3 hours.
  • For inserts (whether PCR products or fragments) run entire reaction on a gel in 2 lanes and extract DNA using Sigma Gel Extraction Kit.
  • For vectors use a standard ethanol precipitation to get rid of buffer and resuspend pellet in 20 ul ddH20.


  • Use DNA immediately for a Ligation (different protocol sheet).



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