Team:UNIPV-Pavia/Notebook/Week2Jul

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(July, 7th)
(July, 9th)
 
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<html><a name="week_start"></a></html>
= Week from July 6th, to July 12nd, 2009 =
= Week from July 6th, to July 12nd, 2009 =
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<tr>
<td align="left" width="50%">
<td align="left" width="50%">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul">
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  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul">Previous Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start">Previous Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul">Next Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul#week_start">Next Week</a>
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  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
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== <html><font class="dayw_style">July, 6th</font></html> ==
== <html><font class="dayw_style">July, 6th</font></html> ==
-
*Digestion for:
+
*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.
 +
 
 +
 
 +
*We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:
{|cellpadding="20"
{|cellpadding="20"
|R0011(E-X)
|R0011(E-X)
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*We stored R0011(E-X) DNA at -20°C.
*We stored R0011(E-X) DNA at -20°C.
-
*We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow an overnight culture (37°C, 220 rpm).
+
*We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).
 +
 
 +
 
 +
 
''Preparation of experiment with Tecan F200''
''Preparation of experiment with Tecan F200''
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*Digestion for BOL1(E-S)(X2).
*Digestion for BOL1(E-S)(X2).
-
*Gel run/cut/purification.
+
*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).
-
*Ligation:
+
*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.
-
**A11 = BOL1(E-S) + R0011(E-X) in pSB1A2
+
-
*We incubated the ligation overnight at 16°C.
+
*We prepared 0.5 l of LB + Amp.
 +
 
 +
*We received sequencing results for:
 +
**T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".
 +
**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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*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).
*We incubated the diluted cultures for 5 hours (37°C, 220 rpm).
 +
 +
 +
 +
''Experiment with Tecan F200''
''Experiment with Tecan F200''
 +
 +
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/culture dilutions TEST 07-07-09.pdf" target="_blank">Download Protocol</a></html>
 +
 +
 +
 +
 +
''Preparation of tomorrow's experiment with Tecan F200''
 +
 +
*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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<div align="right">
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== <html><font class="dayw_style">July, 8th</font></html> ==
== <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.
 +
 +
*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.
 +
 +
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== <html><font class="dayw_style">July, 9th</font></html> ==
== <html><font class="dayw_style">July, 9th</font></html> ==
 +
 +
*We resuspended F2620 BioBrick from iGEM 2009 plates.
 +
 +
*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.
 +
 +
<div align="right">
<div align="right">
[[#top|Top]]
[[#top|Top]]
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== <html><font class="dayw_style">July, 10th</font></html> ==
== <html><font class="dayw_style">July, 10th</font></html> ==
 +
*A11 and F2620 overnight plates showed colonies!
 +
 +
*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.
 +
 +
*We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.
 +
 +
<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
 +
**A11-3
 +
**A11-6
 +
 +
*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.
 +
 +
 +
 +
*We contacted iGEM HQ to request these BioBricks:
 +
{|cellpadding="20"
 +
|K116001
 +
|K116002
 +
|K112405
 +
|-
 +
|P0412
 +
|I746902
 +
|I746903
 +
|-
 +
|K101017
 +
|F2620
 +
|}
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<tr>
<tr>
<td align="left" width="50%">
<td align="left" width="50%">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul">
+
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jul#week_start">
  <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/Week1Jul#week_start">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/Week3Jul#week_start">Next Week</a>
-
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week3Jul">
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  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
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Latest revision as of 23:24, 21 October 2009

EthanolPVanimation.gif

December 2008
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March 2009
M T W T F S S
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2 3 4 5 6 7 8
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30 31
April 2009
M T W T F S S
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13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May 2009
M T W T F S S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June 2009
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July 2009
M T W T F S S
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August 2009
M T W T F S S
1 2
3 4 5 6 7 8 9
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17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September 2009
M T W T F S S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October 2009
M T W T F S S
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November 2009
M T W T F S S
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30

Week from July 6th, to July 12nd, 2009

Previous Week Next Week

July, 6th

  • 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.


  • We already had R0011 and BOL1 purified plasmids stored at -20°C. Digestion for:
R0011(E-X) BOL1(E-S)
  • Gel run/cut and band purification.
  • 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.
  • We stored R0011(E-X) DNA at -20°C.
  • We infected 5 ml of LB + Amp with 8 ul of BOL1 glycerol stock to grow two identical overnight cultures (37°C, 220 rpm).



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.
  • We incubated the inocula overnight at 37°C, 220 rpm.

Top

July, 7th

  • Miniprep for BOL1 (X2).
  • Digestion for BOL1(E-S)(X2).
  • 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).
  • 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.
  • We prepared 0.5 l of LB + Amp.
  • We received sequencing results for:
    • T9002 - long part, but SEQUENCE CONFIRMED! while MIT results were "INCONSISTENT".
    • A10 - results showed a good chromatogram, but K117000 had not been correctly ligated. We will repeat this ligation one of the next weeks.


Preparation of experiment with Tecan F200

  • We diluted 1:100 the overnight cultures of A1, B0030 and J23100.
  • We incubated the diluted cultures for 5 hours (37°C, 220 rpm).



Experiment with Tecan F200



Preparation of tomorrow's experiment with Tecan F200

  • 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.


Top

July, 8th

  • 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.
  • 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.


Top

July, 9th

  • We resuspended F2620 BioBrick from iGEM 2009 plates.
  • 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.


Top

July, 10th

  • A11 and F2620 overnight plates showed colonies!
  • 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.
  • We also picked a colony from F2620 to infect 1 ml of LB + Amp. We incubated this inoculum with the others.

Colony PCR for A11 plate: all the colonies showed the correct plasmid. We kept the 1st, 3rd and 6th colonies.

  • 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
    • A11-3
    • A11-6
  • 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.


  • We contacted iGEM HQ to request these BioBricks:
K116001 K116002 K112405
P0412 I746902 I746903
K101017 F2620

Top



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