Team:Bologna/Lab-Notebook

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HOME TEAM PROJECT MODELING WETLAB LAB-NOTEBOOK DRY LAB 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

Up

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.MARKER
4.TRANS 7
5.PSB1A2

CIS, TRANS 4, MARKER, TRANS 7, PSB1A2


Up

Week 3: from 08/3/09 to 08/7/09

  • Amplification of CIS, TRANS 4 and TRANS 7 using PSB1A2. Inoculation and digestion.
  • Unexpeted results from the gel run in all three sequences (3000, 2200, 1000 bp instead of 2100, 2000, 100 bp):


Digestion of CIS, TRANS 4, TRANS 7 with unexpected 1000 bp insert


  • Done the ligation again whit the same bad results.
  • Done some test fluorescence measurements using GFP in plasmids with different copy number.


Up

Week 4: from 08/10/09 to 08/14/09

HOLIDAY!!!


Up

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 Bmr-genomics in Padova for the dna sequencing.


Up

Week 6: from 08/24/09 to 08/28/09

Bad news for our team:

  • Re-Annealing of our sequences and after have done the PRC-Colony we discovered that the colonies were green. Probably due to a GFP contamination. The DNA sequencing coming from Padova confirmed our theory: in the 1000 bp strand there was the presence of GFP protein and no sign of our CIS or Trans sequences.


Up

Week 7: from 08/31/09 to 09/04/09

  • Done some experiments (trasformation and digestion with the same plasmid, PSB1A2, using different concentration of annealed) to confirm the Gfp contamination. All the gel runs have a 1000 bp strand.
  • Investigation to discover where is the contamination.
  • Starting use a new plasmid: PSB4A3. Trasfomation and digestion with the CIS sequence. From the gel run came out a 400 bp insert that could be correct (CIS+Primer=100bp+270bp=370bp):


PCR Colony: CIS using PSB4A3 in the first well, CIS using PSB3K3 in the others.


  • The same attempt with TRANS 4 and TRANS 7 didn't give us any concrete resulst.


Up