Team:CU-Boulder/Project/Kit/Prototype

All the protocols on this page have been extensively tested to verify which parts matter and which don't. Your results may vary but please try this before changing things.

To begin with create chemically competent cell using Chemically Competent Cells creation protocol.

Next these cells should be transformed using Chemical Transformations Protocol

These cells should then be tested using the Transformation Efficiency Kit that came with your iGEM kit.

After this the restriction enzymes need to be resuspended from the wells. Resuspend your part in 10 microliters of water. iGEM has instructions on exactly how the parts should be resuspended.

Once the restriction enzymes are resuspended they can be transformed using Chemical Transformations Protocol

After these are grown overnight they need to have an overnight culture prepared. Pick a single colony off your plates and grow it in 5mL of LB with the appropriate antibiotic resistance. Remember to do a negative control where you have a vial of LB with antibiotics but no colony to make sure nothing has gone wrong. Overall it is best to create several cultures of 5 mL each of one colony each to increase success and to have more material to work with. DO NOT GROW over the weekend. That is far too long for 5mL and the bacteria will enter into a dieing phase and destroy the proteins you need.

After this has grown overnight it needs to be miniprepped using Protein Miniprep Protocol. It is important that a normal miniprep procedure is not used since that destroys proteins. This step requires being very careful. If you are too rough with the sample the genomic DNA will contaminate your results.

The results of the miniprep has your restriction enzyme. They can then be used in a digest using Digest Lysate Protocol

Once the digestion is complete use Ligation Protocol to ligate your parts together and create a new part. For backbone we suggest doing gel purification instead of PCR.

You can now transform your new part using Chemical Transformations Protocol.