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Team:Bologna/Lab-Notebook
From 2009.igem.org
| HOME | PROJECT | TEAM | SOFTWARE | MODELING | LAB-NOTEBOOK | SUBMITTED PARTS | HUMAN PRACTICE |
|---|
Contents |
Week 1: from 07/20/09 to 07/24/09
Trasfomation of PSB1K3, PSB3K3, PSB1A2, PSB4A3(2007). Only the last one seems to work. We also use PSB1A2.
Trasformation and Amplification of A, B, C:
A) PSB3K3(LMC) + 1429 + RBS + LACI
B) PSB1A2(HC) + 2547 + Ox + RBS + GFP + T
C) PSB3K3(LMC) + 1429 + RBS + GFP + T
Working (using DH5alfa) on:
A + B ---> Evaluation GFP fluorescence imaging comparing with the rRBS one
B ---> Evaluation GFP fluorescence imaging
C ---> Evaluation GFP fluorescence imaging
B+C ---> Evaluation ΣGFP fluorescence imaging
Week 2: from 07/27/09 to 07/31/09
Sequences suspension to have 100 μM conc. (EP digestion and trasformation in a HCN plasmid with AMP)
| DNA sequences | Plasmid |
|---|---|
| H2O mQ == 4.5 μM | H2O mQ == 19.5 μM |
| Sequence == 20 μM | DNA (miniprep) == 5 μM |
| Buffer == 3 μM | Buffer == 3 μM |
| BSA == 0.5 μM | BSA == 0.5 μM |
| EcoRI enzyme == 1 μM | EcoRI enzyme == 1 μM |
| Pst1 enzyme == 1 μM | Pst1 enzyme == 1 μM |
| TOT: 30 μM | TOT: 30 μM |
Gel Run:
1.CIS
2.TRANS 4
3.TRANS 7
4.PSB1A2
(Figure)
Week 3: from 08/3/09 to 08/7/09
Amplification of CIS, TRANS 4 and TRANS 7 using PSB1A2. Inoculation. Unexpeted results from the gel run in all three sequences (3000, 2200, 1000 bp instead of 2100, 2000, 100 bp). Done the ligation again whit the same bad results. Done some test fluorescence measurements using GFP in plasmids with different copy number.
Week 4: from 08/10/09 to 08/14/09
HOLIDAY!!
Week 5: from 08/17/09 to 08/21/09
Done again the amplifications of CIS, TRANS 4 and TRANS 7 using PSB1A2 to undestand which is the problem we have had during the third week. Achieved the same wrong results. We decided to sequence the 1000 bp strand to understand what it really is. We sent our material to the University of Pavia for the dna sequencing.
