Team:UNIPV-Pavia/Notebook/Week12

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(Difference between revisions)
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*Single colonies plates for:
*Single colonies plates for:
-
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006
+
**B0030-C0061-B1006-'''R0062'''-B0030-E0040-B1006 ("Lig.b")
-
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006
+
**B0030-C0078-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.c")
-
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006
+
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006 ("Lig.d")
'''08/5/08'''
'''08/5/08'''
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*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), b(single colonies)(6 colonies), c(single colonies)(6 colonies), 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:
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**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2)
**B0030-C0061-B0030-C0079-B1006-'''R0079'''-B0030-E0040-B1006(2)
-
*We cut these 6 plasmids E-P (5 µl of DNA in a final reaction volume of 20 µl).
+
*We cut these 6 plasmids and Lig.b() 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 (6), Lig.c (5), Lig.d (2)]]
 +
|}
-
*Run for digested plasmids. Gel results:
+
*Gel results:
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive

Revision as of 13:38, 2 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 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 (2nd colony, but it was not pure. We decided to prepare single colonies plate for b)
    • J23100-B0030-C0012-B1006-R0010 (1st colony)

08/4/08

  • Plasmid digestion for:
J23100-B0030-C0040-B1006 (E-S) 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).
  • 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), b(single colonies)(6 colonies), c(single colonies)(6 colonies), 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() 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 (6), Lig.c (5), Lig.d (2)
  • Gel results:
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(1) OK
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(4) False positive
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(6) OK
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006(7) False positive
    • B0030-C0078-B1006-R0079-B0030-E0040-B1006(5) OK
    • B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006(2) OK

08/7/08

  • 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)
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