Team:UNIPV-Pavia/Calendar/September/settimana2

From 2010.igem.org

(Difference between revisions)
(September, 10th)
(September, 10th)
Line 247: Line 247:
|    I51-3  ||  Vector/Screening || 25 ||  1  ||  19,5  ||  1 NheI  || 1 NheI  || 2,5 T
|    I51-3  ||  Vector/Screening || 25 ||  1  ||  19,5  ||  1 NheI  || 1 NheI  || 2,5 T
|}
|}
 +
 +
----
 +
Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and streak the culture spots. Plate were incubated ON at 43°C.
 +
 +
<div align="right"><small>[[#indice|^top]]</small></div>
<div align="right"><small>[[#indice|^top]]</small></div>

Revision as of 14:15, 10 September 2010


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.

MiniPreps of I44-1:5 and I51-1:5 were performed with these results:

Culture Quantifications (ng/ul)
I44-1 11,1
I44-2 9,9
I44-3 12,3
I44-4 11,4
I44-5 11,4
I51-1 13,6
I51-2 19,4
I51-3 16,5
I51-4 13,3
I51-5 21,8

Probably was used the ethanol buffer without adding before the ethanol, during the wash step and so the quantifications were so low.

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

Gel screening shows that all the constructs were ok, so we decided to choose I44-2 and I51-3.

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.


Trasformation of I42 and I43 in MC008 and MG008 strains, strain DB3.1 without DNA and pSB1C3 RFP in DB3.1 strain. MG42, MC43, MC42, MC43 were plated on LB+Cm34 plates instead DB3.1 on LB+Cm12.5 plates. Integration protocol's first day were performed.


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

Screening digestion of I44-2 and I51-3.

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer (ul)
I44-2 Vector/Screening 25 1,5 19 1 EcoRI 1 PstII 2,5 H
I44-2 Vector/Screening 25 1,5 19 1 EcoRI 1 HindIII 2,5 B
I44-2 Vector/Screening 25 1,5 19 1 NheI 1 NheI 2,5 T
I51-3 Vector/Screening 25 1 19,5 1 EcoRI 1 PstII 2,5 H
I51-3 Vector/Screening 25 1 19,5 1 EcoRI 1 Hind 2,5 B
I51-3 Vector/Screening 25 1 19,5 1 NheI 1 NheI 2,5 T

Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and streak the culture spots. Plate were incubated ON at 43°C.


September, 11th