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- | = Week from July 6th, to July 12nd, 2009 = | + | = Week from July 13rd, to July 19th, 2009 = |
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| <table width="100%"> | | <table width="100%"> |
| <tr> | | <tr> |
| <td align="left" width="50%"> | | <td align="left" width="50%"> |
- | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul"> | + | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jul"> |
| <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/> | | <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/> |
| </a> | | </a> |
- | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul">Previous Week</a> | + | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jul">Previous Week</a> |
| </td> | | </td> |
| <td align="right" width="50%"> | | <td align="right" width="50%"> |
- | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul">Next Week</a> | + | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul">Next Week</a> |
- | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul"> | + | <a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4Jul"> |
| <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/> | | <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/> |
| </a> | | </a> |
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- | == <html><font class="dayw_style">July, 6th</font></html> == | + | == <html><font class="dayw_style">July, 13rd</font></html> == |
- | | + | |
- | *We planned to dedicate this week to the assembly of an IPTG/lactose->PoPS device in order to have a lactose sensor for our project, but also a useful inducible system for general purpose applications.
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- | | + | |
- | | + | |
- | *We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:
| + | |
- | {|cellpadding="20"
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- | |R0011(E-X)
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- | |BOL1(E-S)
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- | |}
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- | | + | |
- | *Gel run/cut and band purification.
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- | | + | |
- | *The purified DNA gave a good result at Nanodrop, but we decided to perform the entire work on BOL1 again because we were not sure that we had extracted a pure band.
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- | | + | |
- | *We stored R0011(E-X) DNA at -20°C.
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- | | + | |
- | *We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).
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- | | + | |
- | | + | |
- | | + | |
- | | + | |
- | ''Preparation of experiment with Tecan F200''
| + | |
- | | + | |
- | *We picked a colony of B0030 plate (stored at +4°C) and infected 5 ml of LB + Amp.
| + | |
- | | + | |
- | *We also infected 5 ml of LB + Amp with 10 ul of A1 and J23100 glycerol stocks.
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- | | + | |
- | *We incubated the inocula overnight at 37°C, 220 rpm.
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| </div> | | </div> |
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- | == <html><font class="dayw_style">July, 7th</font></html> ==
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- |
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- | *Miniprep for BOL1 (X2).
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- |
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- | *Digestion for BOL1(E-S)(X2).
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- |
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- | *Gel run/cut/purification: none of the two BOL1 samples gave a good results in DNA quantification...we decided to cut the miniprepped plasmids again, but this time through an overnight digestion (both plasmids E-S).
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- |
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- | *In case of failure, we also infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture at 37°C, 220 rpm.
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- |
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- | *We prepared 0.5 l of LB + Amp.
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- |
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- | *We received sequencing results for:
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- | **T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".
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- | **A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.
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- |
| |
- |
| |
- |
| |
- | ''Preparation of experiment with Tecan F200''
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- |
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- | *We diluted 1:100 the overnight cultures of A1, B0030 and J23100.
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- |
| |
- | *We incubated the diluted cultures for 5 hours (37°C, 220 rpm).
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- |
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- |
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- |
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- |
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- |
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- | ''Experiment with Tecan F200''
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- |
| |
- | * <html><u>Description</u></html>
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- | * <html><u>Purpose:</u></html>
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- | * <html><u>Materials & Methods</u></html>
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- | * <html><u>Protocol</u></html>
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- | * <html><u>Results</u></html>
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- |
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- |
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- |
| |
- |
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- | ''Preparation of tomorrow's experiment with Tecan F200''
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- |
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- | *We picked a colony from B0030 native plate (stored ad +4°C) and infected 5 ml of LB + Amp. We incubated the inoculum at 37°C, 220 rpm overnight.
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| | | |
| + | == <html><font class="dayw_style">July, 14th</font></html> == |
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| <div align="right"> | | <div align="right"> |
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| </div> | | </div> |
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- | == <html><font class="dayw_style">July, 8th</font></html> ==
| |
| | | |
- | *Gel run/cut/band purification for BOL1(E-S) overnight digestion. Results: gel showed that the enzymes had cut very well and this time also the purification gave a good quantification at Nanodrop.
| + | == <html><font class="dayw_style">July, 15th</font></html> == |
- | | + | |
- | *We took R0011(E-X) stored ad -20°C and we were ready to ligate;)
| + | |
- | | + | |
- | *Ligation:
| + | |
- | **A11: BOL1(E-S) + R0011(E-X) in pSB1A2
| + | |
- | | + | |
- | *We incubated the ligation overnight at 16°C.
| + | |
- | | + | |
- | | + | |
- | | + | |
- | | + | |
- | ''Preparation of experiment with Tecan F200''
| + | |
- | | + | |
- | *We diluted 1:100 the overnight culture of B0030.
| + | |
- | | + | |
- | | + | |
- | | + | |
- | | + | |
- | ''Experiment with Tecan F200 (continues the next day)''
| + | |
- | | + | |
- | * <html><u>Description</u></html>
| + | |
- | * <html><u>Purpose:</u></html>
| + | |
- | * <html><u>Materials & Methods</u></html>
| + | |
- | * <html><u>Protocol</u></html>
| + | |
- | * <html><u>Results</u></html>
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| </div> | | </div> |
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- | == <html><font class="dayw_style">July, 9th</font></html> ==
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| | | |
- | *We resuspended F2620 BioBrick from iGEM 2009 plates.
| + | == <html><font class="dayw_style">July, 16th</font></html> == |
- | | + | |
- | *We transformed about 40 pg of the overnight ligation and 1 ul of the resuspended F2620 BioBrick. We plated transformed bacteria and incubated the two plates overnight at 37°C.
| + | |
- | | + | |
- | | + | |
- | | + | |
- | | + | |
- | ''End of experiment with Tecan F200 (last measurement)''
| + | |
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- | == <html><font class="dayw_style">July, 10th</font></html> ==
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- |
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- | *A11 and F2620 overnight plates showed colonies!
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- |
| |
- | *Colony PCR for 6 colonies of A11 plate: the picked colonies were inoculated in 1 ml of LB + Amp and incubated at 37°C, 220 rpm waiting for PCR results.
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- |
| |
- | *We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.
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- |
| |
- | <font class='didascalia'>
| |
- | {|align="center"
| |
- | |[[Image:pv_colonypcr_A11.jpg|thumb|300px|left|Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.]]
| |
- | |}
| |
- | </font>
| |
- |
| |
- | *Gel results: all the picked colonies showed the expected amplicon of a correctly ligated plasmid! We decided to prepare a glycerol stock for:
| |
- | **A11-1
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- | **A11-3
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- | **A11-6
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- |
| |
- | *Next week, only A11-1 will be used, but the other colonies will be used in case of failure.
| |
| | | |
- | *We also prepared a glycerol stock for F2620 culture.
| + | == <html><font class="dayw_style">July, 17th</font></html> == |
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| <div align="right"> | | <div align="right"> |