Team:UNIPV-Pavia/Notebook/Week12

From 2008.igem.org

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!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]
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!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]
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!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]
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!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]
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<br><br>
<br><br>
-
==Week 11: 08/4/08 - 08/8/08==
+
==Week 11: 08/4/08 - 08/7/08==
'''08/4/08'''
'''08/4/08'''
Line 48: Line 60:
*Plasmid digestion for:
*Plasmid digestion for:
{|cellpadding="20px"
{|cellpadding="20px"
-
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (E-S)
+
|J23100-B0030-C0040-'''B1006''' (E-S)
-
|BBa_R0040 (E-X)
+
|R0040 (E-X)
|}
|}
*Gel run/cut/gel extraction.
*Gel run/cut/gel extraction.
-
*Ligation: BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040'''. We incubated ligation at 16°C overnight.
+
*Ligation: J23100-B0030-C0040-B1006-'''R0040'''. We incubated ligation at 16°C overnight.
*We had 5 plates to screen with colony PCR:
*We had 5 plates to screen with colony PCR:
-
**BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062-BBa_B0030-BBa_E1010-BBa_B1006 (that we call "a")
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (that we call "a")
-
**BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062'''-BBa_B0030-BBa_E0040-BBa_B1006 (that we call "b")
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (that we call "b")
-
**BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079'''-BBa_B0030-BBa_E0040-BBa_B1006 (that we call "c")
+
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 (that we call "c")
-
**BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0079-BBa_B1006-'''BBa_R0079'''-BBa_B0030-BBa_E0040-BBa_B1006 (that we call "d")
+
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 (that we call "d")
-
**BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040'''
+
**J23100-B0030-C0012-B1006-'''R0010'''
-
*Last week BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' colony PCR gave a bad result. For this reason, we decided to perform colony PCR only for:
+
*Last week J23100-B0030-C0012-B1006-'''R0010''' colony PCR gave a bad result. For this reason, we decided to perform colony PCR only for:
-
**BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062'''-BBa_B0030-BBa_E0040-BBa_B1006 (7 colonies)
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (7 colonies)
-
**BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006-'''BBa_R0010''' (6 colonies)
+
**J23100-B0030-C0012-B1006-'''R0010''' (6 colonies)
{|
{|
-
|[[Image:pv_colonypcr_27_b.jpg|thumb|300px|left|Marker 1Kb, blank, BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' (6 colonies), b (7 colonies)]]
+
|[[Image:pv_colonypcr_27_b.jpg|thumb|370px|left|Marker 1Kb, blank, J23100-B0030-C0012-B1006-'''R0010''' (6 colonies), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (7 colonies)]]
|}
|}
*Gel result:
*Gel result:
-
**b (2nd colony, but it was not pure. We decided to prepare single colonies plate for b)
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (1st colony, but it was not pure. We decided to prepare single colonies plate for it)
-
**BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006-'''BBa_R0010''' (1st colony)
+
**J23100-B0030-C0012-B1006-'''R0010''' (2nd colony)
'''08/4/08'''
'''08/4/08'''
<br>
<br>
-
*Plasmid digestion for:
+
*We transformed J23100-B0030-C0040-B1006-'''R0040''' overnight ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
-
{|cellpadding="20px"
+
-
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (E-S)
+
-
|BBa_R0040 (E-X)
+
-
|}
+
-
*Gel run/cut/gel extraction.
+
*We infected 9 ml of LB + Amp with 30 µl of C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (1st col) (see "Parts" section for our nomenclature).
-
*We infected 9 ml of LB + Amp with 30 µl of BBa_C0062, 12, 22, 30, 27(2nd col), b(1st col)
+
*Single colonies plates for:
 +
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 ("Lig.b")
 +
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.c")
 +
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.d")
-
*Single colonies plates for b, c, d.
+
'''08/5/08'''
-
 
+
-
'''08/4/08'''
+
<br>
<br>
-
*Glycerol stocks/miniprep for BBa_C0062, 12, 22, 30, 27(2), b(1).
+
*Glycerol stocks/miniprep for C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006(1).
-
*We sent BBa_C0062, 12, 22 and 30 to Primm for sequencing: all these parts contain BBa_C0062.
+
*We sent C0062, Lig.12, Lig.22 and Lig.30 purified plasmids to Primm for sequencing: all these parts contain BBa_C0062.
-
*We transformed/plated 28.
+
*We transformed/plated J23100-B0030-C0040-B1006-'''R0040''' overnight ligation.
-
*Colony PCR for a(7 colonies), b(single colonies)(6 colonies), c(single colonies)(6 colonies), d(single colonies)(6 colonies).
+
*Colony PCR for a (7 colonies), Lig.b(single colonies)(6 colonies), Lig.c(single colonies)(6 colonies), Lig.d(single colonies)(6 colonies).
 +
 
 +
{|
 +
|[[Image:pv_colonypcr_abcd.jpg|thumb|370px|left|Marker 1Kb, blank, Lig.a (7 colonies), Lig.b (6 colonies), Marker 1Kb, Lig.c (6 colonies), Lig.d (6 colonies)]]
 +
|}
*Gel results were not so clear: the length of some fragments was not expected and there were some contaminants. Maybe those parts were too long for our PCR reaction. We decided to grow 9 ml cultures for some of those colonies, to extract plasmids, to cut them and to check their length in a new run. We chose:
*Gel results were not so clear: the length of some fragments was not expected and there were some contaminants. Maybe those parts were too long for our PCR reaction. We decided to grow 9 ml cultures for some of those colonies, to extract plasmids, to cut them and to check their length in a new run. We chose:
-
**a (1, 4, 6, 7)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (1, 4, 6, 7)
-
**b (no colony was chosen: we already had them and this run didn't show any 100% pure colony)
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 (no colony was chosen: we already had them and this run didn't show any 100% pure colony)
-
**c (5)
+
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 (5)
-
**d (2)
+
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 (2)
-
'''08/4/08'''
+
'''08/6/08'''
<br>
<br>
-
*Single colonies plate for 28, because where were too many bacteria on its plate.
+
*Single colonies plate for J23100-B0030-C0040-B1006-'''R0040''', because where were too many bacteria on its plate.
*Glycerol stocks/miniprep for:
*Glycerol stocks/miniprep for:
-
**a(1)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1)
-
**a(4)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4)
-
**a(6)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6)
-
**a(7)
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7)
-
**c(5)
+
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006(5)
-
**d(2)
+
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2)
-
*We cut these 6 plasmids E-P (5 µl)
+
*We cut these 6 plasmids and Lig.b in (E-P) (5 µl of DNA in a final reaction volume of 20 µl).
 +
 
 +
*Run for digested plasmids.
 +
 
 +
{|
 +
|[[Image:pv_colonypcr_aaaabcd.jpg|thumb|370px|left|Marker 1Kb, Lig.a (1), Lig.a (4), Lig.a (6), Lig.a (7), Lig.b (1), Lig.c (5), Lig.d (2)]]
 +
|}
 +
 
 +
*Gel results:
 +
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK
 +
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive
 +
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6) OK
 +
**(Lig.a) B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7) False positive
 +
**(Lig.b) B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006(1) OK
 +
**(Lig.c) B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006(5) OK
 +
**(Lig.d) B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2) OK
 +
 
 +
*We 9 ml of LB + Amp with 30 ul of Lig.b(1) and Lig.c(5) to perform tests.
 +
 
 +
'''08/7/08'''
 +
<br>
 +
*Qualitative fluorescence tests for Lig.b and Lig.c. Results are shown in The Project section (Experiments).
 +
 
 +
*Colony PCR for J23100-B0030-C0040-B1006-'''R0040''' single colonies plate (screening on 6 colonies).
 +
 
 +
*Gel results: all screened colonies were negative...
 +
{|
 +
|[[Image:pv_colonypcr_28_allnegatives.jpg|thumb|300px|left|Marker 1Kb, blank, J23100-B0030-C0040-B1006-'''R0040''' (6 colonies)]]
 +
|}

Latest revision as of 21:27, 26 October 2008


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Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14
Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21
Week 22 Week 23 Week 24



Week 11: 08/4/08 - 08/7/08

08/4/08

  • Plasmid digestion for:
J23100-B0030-C0040-B1006 (E-S) R0040 (E-X)
  • Gel run/cut/gel extraction.
  • Ligation: J23100-B0030-C0040-B1006-R0040. We incubated ligation at 16°C overnight.
  • We had 5 plates to screen with colony PCR:
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (that we call "a")
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 (that we call "b")
    • B0030-C0078-B1006-R0079-B0030-E0040-B1006 (that we call "c")
    • B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006 (that we call "d")
    • J23100-B0030-C0012-B1006-R0010
  • Last week J23100-B0030-C0012-B1006-R0010 colony PCR gave a bad result. For this reason, we decided to perform colony PCR only for:
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 (7 colonies)
    • J23100-B0030-C0012-B1006-R0010 (6 colonies)
Marker 1Kb, blank, J23100-B0030-C0012-B1006-R0010 (6 colonies), B0030-C0061-B1006-R0062-B0030-E0040-B1006 (7 colonies)
  • Gel result:
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 (1st colony, but it was not pure. We decided to prepare single colonies plate for it)
    • J23100-B0030-C0012-B1006-R0010 (2nd colony)

08/4/08

  • We transformed J23100-B0030-C0040-B1006-R0040 overnight ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
  • We infected 9 ml of LB + Amp with 30 µl of C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-R0062-B0030-E0040-B1006 (1st col) (see "Parts" section for our nomenclature).
  • Single colonies plates for:
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 ("Lig.b")
    • B0030-C0078-B1006-R0079-B0030-E0040-B1006 ("Lig.c")
    • B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006 ("Lig.d")

08/5/08

  • Glycerol stocks/miniprep for C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-R0062-B0030-E0040-B1006(1).
  • We sent C0062, Lig.12, Lig.22 and Lig.30 purified plasmids to Primm for sequencing: all these parts contain BBa_C0062.
  • We transformed/plated J23100-B0030-C0040-B1006-R0040 overnight ligation.
  • Colony PCR for a (7 colonies), Lig.b(single colonies)(6 colonies), Lig.c(single colonies)(6 colonies), Lig.d(single colonies)(6 colonies).
Marker 1Kb, blank, Lig.a (7 colonies), Lig.b (6 colonies), Marker 1Kb, Lig.c (6 colonies), Lig.d (6 colonies)
  • Gel results were not so clear: the length of some fragments was not expected and there were some contaminants. Maybe those parts were too long for our PCR reaction. We decided to grow 9 ml cultures for some of those colonies, to extract plasmids, to cut them and to check their length in a new run. We chose:
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (1, 4, 6, 7)
    • B0030-C0061-B1006-R0062-B0030-E0040-B1006 (no colony was chosen: we already had them and this run didn't show any 100% pure colony)
    • B0030-C0078-B1006-R0079-B0030-E0040-B1006 (5)
    • B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006 (2)

08/6/08

  • Single colonies plate for J23100-B0030-C0040-B1006-R0040, because where were too many bacteria on its plate.
  • Glycerol stocks/miniprep for:
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7)
    • B0030-C0078-B1006-R0079-B0030-E0040-B1006(5)
    • B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006(2)
  • We cut these 6 plasmids and Lig.b in (E-P) (5 µl of DNA in a final reaction volume of 20 µl).
  • Run for digested plasmids.
Marker 1Kb, Lig.a (1), Lig.a (4), Lig.a (6), Lig.a (7), Lig.b (1), Lig.c (5), Lig.d (2)
  • Gel results:
    • (Lig.a) B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK
    • (Lig.a) B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive
    • (Lig.a) B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6) OK
    • (Lig.a) B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7) False positive
    • (Lig.b) B0030-C0061-B1006-R0062-B0030-E0040-B1006(1) OK
    • (Lig.c) B0030-C0078-B1006-R0079-B0030-E0040-B1006(5) OK
    • (Lig.d) B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006(2) OK
  • We 9 ml of LB + Amp with 30 ul of Lig.b(1) and Lig.c(5) to perform tests.

08/7/08

  • Qualitative fluorescence tests for Lig.b and Lig.c. Results are shown in The Project section (Experiments).
  • Colony PCR for J23100-B0030-C0040-B1006-R0040 single colonies plate (screening on 6 colonies).
  • Gel results: all screened colonies were negative...
Marker 1Kb, blank, J23100-B0030-C0040-B1006-R0040 (6 colonies)
Retrieved from "http://2008.igem.org/Team:UNIPV-Pavia/Notebook/Week12"