Team:UNIPV-Pavia/Calendar/September/settimana2
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(→September, 10th) |
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Glycerol stocks of MG43, MC42, MC43. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures. | 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/ | + | 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/measuredOD)*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. | 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. | ||
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Trasformation of I42 and I43 in MC008 and MG008 strains, strain DB3.1 without DNA and pSB1C3 RFP in DB3.1 strain. | 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. | MG42, MC43, MC42, MC43 were plated on LB+Cm34 plates instead DB3.1 on LB+Cm12.5 plates. | ||
- | Integration protocol | + | Integration protocol first day were performed. |
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==September, 8th== | ==September, 8th== | ||
+ | |||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test8settembre|Tecan Test]]</font> | ||
+ | |||
+ | ---- | ||
+ | |||
Inoculum of: | Inoculum of: | ||
*I31 | *I31 | ||
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*I36 | *I36 | ||
*I37 | *I37 | ||
+ | *I38 | ||
*I40 | *I40 | ||
*I41 | *I41 | ||
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*I0(E-X) | *I0(E-X) | ||
for second step of ligation of phasins/intein | for second step of ligation of phasins/intein | ||
- | |||
- | |||
- | |||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
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==September, 9th== | ==September, 9th== | ||
Minipreps were quantified as follows: | Minipreps were quantified as follows: | ||
- | *I31: ng/ul | + | *I20: 113.5 ng/ul |
- | *I32: ng/ul | + | *I31: 76.4 ng/ul |
- | *I35: ng/ul | + | *I32: 85.4 ng/ul |
- | *I36: ng/ul | + | *I35: 77.2 ng/ul |
- | *I37: ng/ul | + | *I36: 83.8 ng/ul |
- | * | + | *I37: 156.5 ng/ul |
- | * | + | *I38: 94.4 ng/ul |
- | * | + | *I40: 556 ng/ul |
+ | *I41: 148.2 ng/ul | ||
They were digested: | They were digested: | ||
+ | *I20: SpeI-PstI | ||
*I31: EcoRI-XbaI | *I31: EcoRI-XbaI | ||
- | *I32: E- | + | *I32: E-S |
- | *I35: S- | + | *I35: S-P |
*I36: E-S | *I36: E-S | ||
*I37: E-X; X-P | *I37: E-X; X-P | ||
+ | *I38: E-S | ||
*I40: E-S | *I40: E-S | ||
*I41: E-X | *I41: E-X | ||
- | + | ||
---- | ---- | ||
- | MG42 and MG43 | + | MG42 and MG43 showed no colonies: probably the reason was a mistake on the choice of the MG008 strain tubes during the trasformations. |
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==September, 10th== | ==September, 10th== | ||
+ | |||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test10settembre_bis|Tecan Test]]</font> | ||
+ | |||
+ | ---- | ||
Screening digestion of I44-2 and I51-3. | Screening digestion of I44-2 and I51-3. | ||
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|} | |} | ||
- | [[Image:UNIPV10_10_09_10_Screening_I44(E-P)_I44(E-H)_I44(N-N)_I51(E-P)_I51(E-H)_I51(N-N).jpg|thumb| | + | [[Image:UNIPV10_10_09_10_Screening_I44(E-P)_I44(E-H)_I44(N-N)_I51(E-P)_I51(E-H)_I51(N-N).jpg|thumb|300px|center| Check for I44 and I51.]] |
- | |||
Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and the culture spots were streaked on the plates. Plates were incubated ON at 43°C. | Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and the culture spots were streaked on the plates. Plates were incubated ON at 43°C. | ||
+ | ---- | ||
+ | Gel run/cut for samples of previous day. Gel extraction showed the following quantifications: | ||
+ | *I31 (E-X): 21.2 ng/ul | ||
+ | *I32 (E-S): 11.3 ng/ul | ||
+ | *I35 (S-P): 24.6 ng/ul | ||
+ | *I36 (E-S): 12.1 ng/ul | ||
+ | *I37 (E-X): 26 ng/ul | ||
+ | *I37 (X-P): 12.2 ng/ul | ||
+ | *I38 (E-S): 10.8 ng/ul | ||
+ | *I40 (E-S): 16.3 ng/ul | ||
+ | *I41 (E-X): 24.4 ng/ul | ||
+ | *I20 (S-P): 30.3 ng/ul | ||
+ | |||
+ | Digestions were ligated in order to create: | ||
+ | *I52: I35(S-P)+I37(X-P) | ||
+ | *I53: I32(E-S)+I37(E-X) | ||
+ | *I54: I36(E-S)+I37(E-X) | ||
+ | *I55: I38(E-S)+I20(E-X) | ||
+ | *I56: I26(E-S)+I41(E-X) | ||
+ | *I57: I40(E-S)+I0(E-X) | ||
+ | *I58: I38(E-S)+I31(E-X) | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==September, 11th== | ==September, 11th== | ||
+ | MC42 and MC43 plates were transfered at -4°C. | ||
+ | |||
+ | I42 and I43 have been transformed in MG008 strain, plated on LB+Cm34 plates and incubated at 30°C for 48 hours. | ||
+ | |||
+ | |||
+ | |||
+ | |||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> |
Latest revision as of 19:14, 27 October 2010
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