Team:UNIPV-Pavia/Calendar/September/settimana5
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- | <html><p align="center"><font size="4"><b>SEPTEMBER: WEEK | + | <html><p align="center"><font size="4"><b>SEPTEMBER: WEEK 5</b></font></p></html><hr><br> |
<html><a name="indice"/></html> | <html><a name="indice"/></html> | ||
==September, 27th== | ==September, 27th== | ||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test27settembre|Tecan Test]]</font> | ||
- | + | ---- | |
- | + | MiniPrep and E-P digest for I62-a, I62-b, I62-c, I62-d, I62-e, I62-f | |
- | + | DNA PCR for MyYeast 1-2-3 | |
- | + | Gel run | |
+ | [[Image:UNIPV10_27_09_2010_I62_screen.jpg|thumb|200px|center|I62 and MyYeast screening.]] | ||
- | |||
- | Protein electrophoresis for phasins | + | I62-e was positive, so we made glycerol stock and stored it at -80°C. |
+ | |||
+ | |||
+ | Protein electrophoresis for phasins. | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==September, 28th== | ==September, 28th== | ||
- | + | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test28settembre|Tecan Test]]</font> | |
- | < | + | |
- | + | ||
- | + | ||
- | + | ||
---- | ---- | ||
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*<partinfo>pSB4C5</partinfo> (DNA already available) (E-P) | *<partinfo>pSB4C5</partinfo> (DNA already available) (E-P) | ||
- | Gel run for all digested parts and | + | |
+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
+ | |||
+ | ==September, 29th== | ||
+ | |||
+ | Colony PCR for 6 newly picked colonies. | ||
+ | [[Image:UNIPV10_29_09_2010_I55_screen.jpg|thumb|200px|center|I55 colony PCR screening.]] | ||
+ | No positives nor this time. | ||
+ | ---- | ||
+ | |||
+ | |||
+ | Gel run for all digested parts | ||
+ | [[Image:UNIPV10_29_09_2010_digestions.jpg|thumb|200px|center|Digestions.]] | ||
+ | and cut/gel purification for: | ||
*4C5 (E-P): 19,3 ng/ul | *4C5 (E-P): 19,3 ng/ul | ||
*My Yeast 3 (E-S): 20,8 ng/ul | *My Yeast 3 (E-S): 20,8 ng/ul | ||
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*<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul | *<partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul | ||
*<partinfo>pSB1C3</partinfo>: 10 ng/ul | *<partinfo>pSB1C3</partinfo>: 10 ng/ul | ||
- | |||
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|} | |} | ||
+ | ---- | ||
+ | |||
+ | Inoculum of MC43, MG43 in 5ml LB with chloramphenicol concentration of 12.5 ug/ml. | ||
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==September, 30th== | ==September, 30th== | ||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test30settembre|Tecan Test]]</font> | ||
+ | |||
+ | ---- | ||
+ | |||
Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate. | Transformation of I73 (1ul) into ''E. coli'' TOP10. It was plated on LB+Amp 100 ug/ml agar plate. | ||
+ | |||
+ | Trasformation of: | ||
+ | *I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp | ||
+ | *I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp | ||
+ | *I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5 | ||
+ | |||
+ | All plates were incubated at 37°C ON | ||
+ | |||
Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes: | Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes: | ||
*I20: (E-X) | *I20: (E-X) | ||
- | Gel run/cut | + | Gel run/cut/extraction/quantification |
- | + | [[Image:UNIPV10_30_09_2010_I20_E_X.jpg|thumb|70px|center|I20 E-X digest.]] | |
- | + | I20 E-X: 13,4 ng/ul | |
We repeated I55 ligation using newly digested vector and already digested I38 (E-S) | We repeated I55 ligation using newly digested vector and already digested I38 (E-S) | ||
+ | |||
+ | I55: I38 (E-S) + I20 (E-X) | ||
Inoculum into 5ml LB+Amp of: | Inoculum into 5ml LB+Amp of: | ||
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*I60 | *I60 | ||
*INTEIN | *INTEIN | ||
- | *3x <partinfo>pSB1C3</partinfo> | + | *3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on) |
Cultures were let grow ON at 37°C, 220 rpm. | Cultures were let grow ON at 37°C, 220 rpm. | ||
---- | ---- | ||
+ | Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!! | ||
+ | Inoculum of: | ||
+ | |||
+ | *S1 in 500ul M9+Cm 12,5 | ||
+ | *I47-S1 in 500ul M9+Amp+Cm 12,5 | ||
+ | *GFP-S1 in 500ul M9+Amp+Cm 12,5 | ||
+ | *RBS32 in 500ul M9+Amp | ||
+ | |||
+ | Cultures were grown ON at 37°C 220 rpm. | ||
+ | |||
+ | Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added. | ||
+ | |||
+ | ---- | ||
+ | In order to excise the chloramphenicol resistance cassette from the integrants in E.coli genome we transformed the cells with a plasmid (pCP20 plasmid) carrying the gene encoding for the flippase protein. This protein is able to cut fragment between two FRT flanking sequences. pCP20 was transformed in MC43-a and MG43-a and plated on LB+Amp agar plate. | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==October, 1st== | ==October, 1st== | ||
+ | |||
+ | <font size=4>[[Team:UNIPV-Pavia/Material Methods/Measurements/Tecan/test1ottobre|Tecan Test]]</font> | ||
+ | |||
+ | ---- | ||
+ | |||
+ | This morning, we checked tranformed plates: | ||
+ | |||
+ | *I72 showed NO colony!! :/ we will repeat this ligation next week | ||
+ | *I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening. | ||
+ | *I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | Transformation of I55 into ''E. coli'' DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | Miniprep and Nanodrop quantification for: | ||
+ | *I31: 76,1 ng/ul | ||
+ | *I35: 66,8 ng/ul | ||
+ | *I60: 279,7 ng/ul | ||
+ | (they were also sent sequencing) | ||
+ | *INTEIN: 123,8 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul | ||
+ | *I37: 174,9 ng/ul | ||
+ | |||
+ | Digest: | ||
+ | *<partinfo>pSB1C3</partinfo>: E-P | ||
+ | *<partinfo>pSB1C3</partinfo>: E-S | ||
+ | *<partinfo>pSB1C3</partinfo>: X-P | ||
+ | *INTEIN: E-P | ||
+ | *I60: E-S | ||
+ | *I31: S-P | ||
+ | *I35: E-P | ||
+ | *I35: E-X | ||
+ | *I37: E-X | ||
+ | *I57: E-X | ||
+ | |||
+ | Gel run/cut/extraction/quantification: | ||
+ | {|align="center" | ||
+ | |[[Image:UNIPV10_01_10_2010_digestioni_medio.jpg|thumb|200px|center|Intein, I31, I35, I37, I57, I60 digestions]] || [[Image:UNIPV10_01_10_2010_digestioni_psb1c3.jpg|thumb|150px|center|<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.]] | ||
+ | |} | ||
+ | |||
+ | *I37 (E-X): 20,4 ng/ul | ||
+ | *I57 (E-X): 17,6 ng/ul | ||
+ | *I60 (E-S): 9,5 ng/ul | ||
+ | They were stored at -20°C. | ||
+ | *I31 (E-S): 5,4 ng/ul | ||
+ | *I35 (E-P): 1,0 ng/ul ;-( | ||
+ | *I35 (E-X): 14,4 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul | ||
+ | *<partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul | ||
+ | |||
+ | ON ligation of: | ||
+ | *I74: I31 (E-S) + I35 (E-X) | ||
+ | *INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P) | ||
+ | |||
+ | ---- | ||
+ | |||
+ | Single colony was picked from MC43 and MG43 plates and inoculated in 5 ml of LB and incubated at 37°C, 220 rpm ON. | ||
+ | 12 hours late 5 ul were streaked on non selective LB plates and incubated at 43°C ON in order to ensure the loss of pCP20 plasmid. | ||
+ | |||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> | ||
==October, 2nd== | ==October, 2nd== | ||
+ | |||
+ | PCR from colony for I55 (eleven colonies were picked). | ||
+ | |||
+ | Gel run: | ||
+ | |||
+ | [[Image:UNIPV10_02_10_2010_I55_screening.jpg|thumb|200px|center|I55 colony PCR screening.]] | ||
+ | |||
+ | We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm. | ||
+ | ---- | ||
+ | Miniprep and quantification for I73-1/2/3/4/5/6: | ||
+ | *I73-1: 154 ng/ul | ||
+ | *I73-2: 177 ng/ul | ||
+ | *I73-3: 154,7 ng/ul | ||
+ | *I73-4: 145,5ng/ul | ||
+ | *I73-5: 152,6 ng/ul | ||
+ | *I73-6: 154,2 ng/ul | ||
+ | 1,5 hours E-P digest (for screening) and gel run | ||
+ | |||
+ | [[Image:UNIPV10_02_10_2010_I73_screening.jpg|thumb|200px|center|I70, I71, I73, A9_4C5, A99_4C5 screening.]] | ||
+ | |||
+ | As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C. | ||
+ | |||
+ | ---- | ||
+ | Transformation of I74 and INTEIN_C3 (1ul) in ''E. coli'' TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C. | ||
+ | |||
+ | ---- | ||
+ | Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | 3 colonies each plate were picked from MC43_flip and MG43_flip plates and inoculated in 5 ml of non selective LB. To confirm the loss of the chloramphenicol resistance cassette and of the pCP20 plasmid, each colony was inoculated also in 5 ml of LB+Cm12.5 and 5 ml of LB+Amp. | ||
+ | |||
+ | <div align="right"><small>[[#indice|^top]]</small></div> | ||
+ | |||
+ | ==October, 3rd== | ||
+ | Glycerol stock for: | ||
+ | *I55-1 | ||
+ | *IC-1/2/3/4-4C5 | ||
+ | |||
+ | ---- | ||
+ | Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm. | ||
+ | |||
+ | ---- | ||
+ | Miniprep and quantification of: | ||
+ | *I55-1: 57,4 ng/ul | ||
+ | *I55-1bis: 79,6 ng/ul | ||
+ | |||
+ | I55-1bis was digested E-S for about three hours and than gel run/cut. | ||
+ | |||
+ | ---- | ||
+ | |||
+ | Colony PCR for I74-1..6. | ||
+ | |||
+ | ---- | ||
+ | Gel run for PCR samples and I55 (E-S) and cut for this one. | ||
+ | [[Image:UNIPV10_03_10_2010_I74_screen_I55_cut.jpg|thumb|200px|center|I74 screening and I55 E-S cut.]] | ||
+ | Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day. | ||
+ | |||
+ | Gel extraction and quantification: | ||
+ | *I55 (E-S): 5,0 ng/ul | ||
+ | |||
+ | Ligation of I55 (E-S) with already available DNA: | ||
+ | *I75: I55 (E-S) + I37 (E-X) | ||
+ | *I77: I55 (E-S) + I57 (E-X) | ||
+ | ---- | ||
+ | Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm. | ||
+ | |||
+ | ---- | ||
+ | Each culture of MC43_flip and MG43_flip in selective LB didn't grown, confirming the loss of pCP20 plasmid and of the chloramphenicol resistance cassette integrated into the E.coli genome. | ||
<div align="right"><small>[[#indice|^top]]</small></div> | <div align="right"><small>[[#indice|^top]]</small></div> |
Latest revision as of 18:36, 27 October 2010
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