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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)
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| I44-1 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I44-2 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I44-3 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I44-4 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I44-5 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I51-1 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I51-2 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I51-3 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I51-4 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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| I51-5 | Screening | 25 | 21,5 | 0 | 0,5 EcoRI | 0,5 HindIII | 2,5
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September, 7th
MiniPrep for:
| Culture | Quantifications (ng/ul)
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| <partinfo>BBa_J23100</partinfo>_4C5 | 11,8
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| <partinfo>BBa_J23101</partinfo>_4C5 | 39,7
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| <partinfo>BBa_J23105</partinfo>_4C5 | 25,4
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| <partinfo>BBa_J23106</partinfo>_4C5 | 29,7
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| <partinfo>BBa_J23110</partinfo>_4C5 | 24,7
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| <partinfo>BBa_J23114</partinfo>_4C5 | 15,4
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| <partinfo>BBa_J23116</partinfo>_4C5 | 20,7
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| <partinfo>BBa_J23118</partinfo>_4C5 | 20,4
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| I7-A | 113,6
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| I8-5-D | 127,6
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| I44-2 (Cm 34) | 63,9
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| I51-3 (Cm 34) | 100,4
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| I44-2 (Cm 12,5) | 68,1
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| I51-3 (Cm 12,5) | 135,4
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| I47 | 74,8
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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
September, 11th
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