Team:Tuebingen/Notebook/Journal/Weekly

Week 26

Week 25

Week 24

Week 23

Week 22

Week 21

Week 20

Week 19

Week 18

Week 17

Week 16

Week 15

Week 14

Week 13

Week 12

Week 11

Week 10

Week 9

Week 8

Week 7

Week 6

Week 5

Week 4

Pfet3 PCRs remainded a huge problem - nothing we tried seemed to work out. Also, we have produced new chemo-competent cells and tried to test the pTUM-vectors by ligation with some of our parts. We also tried to amplify a full fragment of luc (after successful site-directed-mutagenesis). After many experiments we have finally found an ideal protocol for gelelectrophoreses that does not include expensive post-staining: we basically use more loading buffer and make the wells full to overflow!

Week 3

The first thing we did in this week, was a control restriction of last week's ligations. Unfortunately, not one single ligation was successful thus this whole step had to be repeated. Our work with luc (i.e. luc quick-change) continued, additional PCRs went well, and we made plans how to control luc's quality once both restriction sites were eliminated.

This week, we received a package containing pTUM yeast vectors from TU Munich's iGEM Team! We need the pTUM vectors from pTUM100 to pTUM104 in order to add missing BioBrick restriction sites to some of our parts.

Week 2

Further PCRs of Panb1 and Pfet3 were more promising but Pfet3 still made lots of problems because there were only very faint Pfet3 stains on our gels. We transformed pSB1C3 and RFP into E. coli and our first plasmid extractions of these parts went pretty smooth. We yielded pretty good amounts of DNA and after some restriction digests we were able to ligate Panb1 into pSB1C3!

We started out our iGEM project with several unsuccessful PCRs of Pfet3. We also tried restriction digests and ligations of Psuc2, rox1, mig1, and the mPRs into pSB1C3– without positive results. Unfortunately, we had massive problems (smiling marker) with staining our gelelectrophoresis gels thus had to start off our project with trouble-shooting. After some experiments we came to the conclusion that post-staining would be an expensive way to solve our gel problems. Due to better gels we could prove the successful PCRs of Panb1 and Psuc2 and promptly started ligation and transformation of these parts. Initial transformations of mig1 were unsuccessful. However, luc PCRs went very well and we already had highest hopes for this part. Unfortunately, we found two illegal restriction sites (EcoRI and XbaI) inside of luc thus we had to come up with something special: site-directed-mutagenesis (quick-change) using 3 pairs of primers and Pfu-polymerase. Restricitons of Padh1, Psuc2, rox1, mig1, mPR Dr, mPR Xl and RFP (our initial reporter) went rather well but due to missing negative controls in the control gel further steps were not possible this week.