Procedures

From 2006.igem.org

Once this is completed it will be printed and available in the lab. It should include all the details so as to allow someone not familiar with a procedure to carry it out perfectly. Please add steps, comments etc. as you see fit.

Overnight Seeding:

• 1. Prepare desired volume of 1X LB all in one culture tube (calculate 1mL per seeding).
• 2. Add desired antibiotic (1uL per mL for Ampicilin).
• 3. Split the total volume by pipetting 1mL into additional culture tubes until each tube has 1mL.
• 4. Pick colonies and pipet up and down in LB to mix thoroughly.
• 5. Place in S-I overnight.

N.B.: Close tubes only partially to let air in.

Amplification Seeding:

• 1. Prepare desired volume of 1X LB + Antibiotic in culture tubes.
• 2. Pipet 100-200uL of overnight culture into the fresh LB.
• 3. Place in S-I until desired O.D. is reached.

Induction

Plating

Miniprep

Midiprep

Gel Electrophoresis

Gel Purification

Transformation

Ligation

PCR :

Step 1: Take the DNA that is 1.28 ug/uL and make a 1000 fold dilution by taking another tube, adding 1000 uL PCR water and adding 1 uL of DNA. Mix Gently by inverting.

Step 2: The primers arrive in a form that is DRY. Read on the label how many nanograms of DNA there are and add 10X as many uL of EB solution (important: Use the EB solution upstairs that Anette dedicated to PCR primers!!! You don't want to contaminate primers with regular EB) and making a 0.1 ng/uL solution. For example if 26.1 ng DNA, we add 261 uL water. After water addition the extra primers can be stored in the Freezer just like all other DNA.

Spep 3: A PCR tube is taken and the following additions are performed exactly in the following order. There is a P2 pipette available upstais if one has to use it.

• A: 80 uL PCR water
• B: 10 uL PCR buffer
• C: 4 uL dNTP's
• D: 2 uL each Forward and back Primer
• E: 1 uL BSA
• F: 1 uL Taq polymerase (Important Note: Take it only when absolutely ready for it. It is taken out of the fridge and into the portable freezerbox only when the things above have been added. After use it must immediately return to its place in the freezer.
• G: 2 uL DNA, the Diluted solution

Step 4: The PCR tube is placed at the centre of the rack in the PCR machine and program 84 is turned on. Under no circumstances can one attempt to reprogram the machine.