• Home
• Team
• Project
• Parts
• Modelling
• Sponsors
• Lab Book

Project

• Overview
• Choices Rationale
• Characterisation
• Safety
• Judging Comments

Sub-projects

• Metal Intake/Efflux
• Metal Sensing
• Sporulation Tuning
• Population Dynamics
• Stochastic Switch
• Metal Sequester
• Chassis
• Promoter Library

Wet Lab

• Lab Book
• Protocols

Meetings

• Meetings Calendar

Planning

• Timeline
• Ideas

Links

• Helping other teams
• Ethics
• T-Shirts
• iGEM Meetup
• Our City
• Fun and Games
• Sponsors
• Useful Links
• Contact Us
• Press Coverage

Contents 1 Professor Phil Aldridge's Lab Protocols 1.1 Transforming DNA "Phil Style" 1.2 Restriction Digests 1.3 Ligation Reactions

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).

Ligation Reactions

In general

• Before attempting a ligation you should read the information on the back
• Ref: Molecular cloning vol 1 Sambrook et al.
• Solutions required:
  • Digested DNA
  • T4 DNA ligase and buffer

Ligations

• All ligations should have a final volume of 20 ul.
• Your vector DNA should have a concentration of 0.05 ug/ul at the end of all manipulations.
  • N.B. Assume maximum recovery and use your starting concentration!

• All ligations must contain the following:
  • Sterile H2O giving a final volume of 20 ul
  • 2 ul 10x ligation buffer
  • 1 - 16 ul insert (see note)
  • 1 ul 0.05 ug/ul vector DNA
  • 1 ul T4 DNA ligase

• Controls are very important here, especially cut vector with ligase and cut vector without ligase.
• Incubate on the top shelf of a fridge overnight before transforming 10 - 20 ul into appropriate cells.

Note: INSERT DNA

• The general rule is to have excess molar amounts of insert to vector.
• If you cut your insert out of a gel and it is SMALLER than your vector you can take 16 ul of the elution without doing any quantification.
• This also stands for PCR products (as they are almost always smaller than the vector). However, sometimes it is not necessary to elute from a gel.

Page 1.67 (sambrook)

...proportion of transformed bacterial colonies that carry recombinant plasmids. In this case, it is advisable to consider taking steps to reduce the background of colonies carrying nonrecombinant plasmids either by treating the linearized plasmid DNA with phosphatase or by adopting another cloning strategy so that the recombinant plasmid can be constructed by directional cloning.