Team:UNIPV-Pavia/Calendar/September/settimana2

From 2010.igem.org

Revision as of 13:54, 9 September 2010 by Matteo (Talk | contribs)


SEPTEMBER: WEEK 2



September, 6th

Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN experiment of the following day.


  • Inoculum of I44-2 and I51-3 into 5 ml LB+Cm34 to evaluate MiniPrep yields.
  • Inoculum of I44-2 and I51-3 into 5 ml LB+Cm12,5 to evaluate MiniPrep yields and for sequencing.
  • Inoculum for MiniPrep and sequencing of:
    • <partinfo>BBa_J23100</partinfo>_4C5
    • <partinfo>BBa_J23101</partinfo>_4C5
    • <partinfo>BBa_J23105</partinfo>_4C5
    • <partinfo>BBa_J23106</partinfo>_4C5
    • <partinfo>BBa_J23110</partinfo>_4C5
    • <partinfo>BBa_J23114</partinfo>_4C5
    • <partinfo>BBa_J23116</partinfo>_4C5
    • <partinfo>BBa_J23118</partinfo>_4C5
    • I7-A
    • I8-5-D

Inoculum of cultures for TECAN test on self-inducible promoters:

  • I14-1
  • I15-1
  • I16-1
  • I17-1
  • I18-1
  • I19-1
  • I14_4C5-2
  • I15_4C5-2
  • I16_4C5-1
  • I17_4C5-1
  • I18_4C5-1
  • I19_4C5-1
  • I6-3
  • I3-1
  • <partinfo>BBa_B0031</partinfo>

Glycerol stocks of MG43, MC42, MC43. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.

Cultures were diluted in 4 ml final of LB in order to obtain the same lowest OD with the following formula: culture to remove and substitute with LB=FinalVolume-(lowestOD/misuredOD)*FinalVolume. Centrifuge at 4000 rpm, 25°C for 5 min to pellet the culture, discard the surnatant and resuspend the pellet with 1 mL of M9. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.


Liquid PCR was performed on MG43 (3 colonies), MC42 (3 colonies), MC43 (3 colonies), MC1061 (negative control) with the following parameters: T_annealing=52°C, cycles composed by 10 min at 94°C, 30 sec at 94°C, 30 sec at 52°C and 4 min at 72°C . This is the result:

PCR results for MG43 A-B-C, MC42 A-B-C, MC43 A-B-C, MC1061. The last lane is reaction blank.

Digestion of I44_1:5 and I51_1:5 performed as follow:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I44-1 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I44-2 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I44-3 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I44-4 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I44-5 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I51-1 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I51-2 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I51-3 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I51-4 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5
I51-5 Screening 25 21,5 0 0,5 EcoRI 0,5 HindIII 2,5


September, 7th

MiniPrep for:

CultureQuantifications (ng/ul)
<partinfo>BBa_J23100</partinfo>_4C5 11,8
<partinfo>BBa_J23101</partinfo>_4C5 39,7
<partinfo>BBa_J23105</partinfo>_4C5 25,4
<partinfo>BBa_J23106</partinfo>_4C5 29,7
<partinfo>BBa_J23110</partinfo>_4C5 24,7
<partinfo>BBa_J23114</partinfo>_4C5 15,4
<partinfo>BBa_J23116</partinfo>_4C5 20,7
<partinfo>BBa_J23118</partinfo>_4C5 20,4
I7-A 113,6
I8-5-D 127,6
I44-2 (Cm 34) 63,9
I51-3 (Cm 34) 100,4
I44-2 (Cm 12,5) 68,1
I51-3 (Cm 12,5) 135,4
I47 74,8

Cultures for TECAN test were diluted in the morning (h10:20) 1:100 (40ul in 4ml) and let grown at 37°C 220 rpm till they reach the desired OD.

September, 8th

Inoculum of:

  • I31
  • I32
  • I34
  • I35
  • I36
  • I37
  • I40
  • I41
  • I20

into 5 ml LB + Amp; already available:

  • I21(E-S)
  • I26(E-S)
  • I0(E-X)

for second step of ligation of phasins/intein



September, 9th

Minipreps were quantified as follows:

  • I31: ng/ul
  • I32: ng/ul
  • I34: ng/ul
  • I35: ng/ul
  • I36: ng/ul
  • I37: ng/ul
  • I40: ng/ul
  • I41: ng/ul
  • I20: ng/ul

September, 10th

September, 11th