Team:UNIPV-Pavia/Calendar/September/settimana2
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*<partinfo>BBa_B0031</partinfo> | *<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/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. | ||
+ | |||
+ | ---- | ||
+ | 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: | ||
+ | |||
+ | [[Image:UNIPV10_06_09_10_PCR_MG43ABC_MC42ABC_MC43ABC_Cneg_Blank.jpg|thumb|200px|center|PCR results for screening.]] | ||
+ | |||
+ | ---- | ||
+ | MiniPreps of I44-1:5 and I51-1:5 were performed with these results: | ||
+ | |||
+ | {| border="1" align='center' | ||
+ | | ''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: | ||
+ | |||
+ | {| border="1" align='center' | ||
+ | | ''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. | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
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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. | 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 first day were performed. | ||
+ | |||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==September, 8th== | ==September, 8th== | ||
+ | |||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test8settembre|Tecan Test]]</font> | ||
+ | |||
+ | ---- | ||
+ | |||
Inoculum of: | Inoculum of: | ||
*I31 | *I31 | ||
*I32 | *I32 | ||
- | |||
*I35 | *I35 | ||
*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: | ||
- | * | + | *I20: 113.5 ng/ul |
- | * | + | *I31: 76.4 ng/ul |
- | * | + | *I32: 85.4 ng/ul |
- | *I35: ng/ul | + | *I35: 77.2 ng/ul |
- | *I36: ng/ul | + | *I36: 83.8 ng/ul |
- | *I37: ng/ul | + | *I37: 156.5 ng/ul |
- | * | + | *I38: 94.4 ng/ul |
- | * | + | *I40: 556 ng/ul |
- | * | + | *I41: 148.2 ng/ul |
+ | They were digested: | ||
+ | *I20: SpeI-PstI | ||
+ | *I31: EcoRI-XbaI | ||
+ | *I32: E-S | ||
+ | *I35: S-P | ||
+ | *I36: E-S | ||
+ | *I37: E-X; X-P | ||
+ | *I38: E-S | ||
+ | *I40: E-S | ||
+ | *I41: E-X | ||
+ | |||
+ | |||
+ | ---- | ||
+ | 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. | ||
+ | |||
+ | {| border="1" align='center' | ||
+ | | ''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 | ||
+ | |} | ||
+ | |||
+ | [[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. | ||
+ | |||
+ | ---- | ||
+ | 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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