Basic Summary

From 2006.igem.org

Revision as of 03:48, 28 October 2006

Basic Summary: For vectors two plasmids arrived from Spain called Fos-Beta and Jun-Beta. These arrived in the form of cells in agar tubes. They were seeded, midiprepped and confirmed with a restriction digest. They were than digested first with SacII in NEB buffer 4 for 3 hours, than EcoRI and EcoRI buffer were added for another 1h and this was heat deactivated at 65OC for 20. The reason this did not run on a gel was that the part that comes off is too small to recircularize it. For the insert, the template arrived from Michigan. It was called pDsBiFc- bJunYN155- bFosYC155, and had ampicillin resistance. It was transformed, seeded and miniprepped as well as confirmed with a restriction digest. This was than diluted 50 fold to serve as a PCR template. PCR primers with restriction sites at their ends for SacII and EcoRI were ordered. Out of the template plasmid, two different PCR reactions were performed: YFPC (C terminal part of YFP protein) as well as YFPN (N terminal part). (Note: In the plasmid, the YFP protein had already been split in two and each was a fusion protein). After PCR the products were digested with EcoRI, ran on a gel, extracted, and digested with SacII. For the ligation, the YFPC was ligated to the Fos-Beta in a 1:1 mass ratio (100ng each) and the YFPN was ligated to Jun-Beta in 1:1 mass ratio (59ng each). The ligation plates were screened by a method called “cracking” which constitutes of performing a PCR reaction directly from a ligation plate. Almost all colonies of each ligation were positive. They were than seeded, miniprepped, and transformed into BL21 cells. This transformation was seeded and induced with iPTG and viewed under fluorescence microscope, which resulted in adhesion and fluorescence upon adding Fos-Beta-YFPC cells to Jun-Beta-YFPN cells but not each type separately.