Team:UNIPV-Pavia/Notebook/Week12

From 2008.igem.org

(Difference between revisions)
Line 56: Line 56:
*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-C0040-B1006-'''R0040'''
-
*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|300px|left|Marker 1Kb, blank, J23100-B0030-C0040-B1006-'''R0040''' (6 colonies), b (7 colonies)]]
|}
|}
*Gel result:
*Gel result:
**b (2nd colony, but it was not pure. We decided to prepare single colonies plate for b)
**b (2nd colony, but it was not pure. We decided to prepare single colonies plate for b)
-
**BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006-'''BBa_R0010''' (1st colony)
+
**J23100-B0030-C0012-B1006-'''R0010''' (1st colony)
'''08/4/08'''
'''08/4/08'''
Line 87: Line 87:
*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.
-
*We infected 9 ml of LB + Amp with 30 µl of BBa_C0062, 12, 22, 30, 27(2nd col), b(1st col)
+
*We infected 9 ml of LB + Amp with 30 µl of C0062, 12, 22, 30, 27(2nd col), b(1st col) (see "Parts" section for our nomenclature).
*Single colonies plates for b, c, d.
*Single colonies plates for b, c, d.
Line 99: Line 99:
'''08/5/08'''
'''08/5/08'''
<br>
<br>
-
*Glycerol stocks/miniprep for BBa_C0062, 12, 22, 30, 27(2), b(1).
+
*Glycerol stocks/miniprep for C0062, 12, 22, 30, 27(2), b(1).
-
*We sent BBa_C0062, 12, 22 and 30 to Primm for sequencing: all these parts contain BBa_C0062.
+
*We sent C0062, 12, 22 and 30 to Primm for sequencing: all these parts contain BBa_C0062.
*We transformed/plated 28.
*We transformed/plated 28.
-
*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).
*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:
Line 140: Line 140:
*Gel results: all screened colonies were negative...
*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), b (7 colonies)]]
 +
|}

Revision as of 10:05, 2 October 2008


Home.jpg Home Unipv logo.jpg The Team And.jpg The Project Safety.jpg Biological Safety Dna.png Parts Submitted to the Registry
Laptop.jpg Dry Lab Pipette.jpg Wet Lab Math.gif Modeling Note.jpg Protocols Notebook.gif Activity Notebook



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-C0040-B1006-R0040
  • 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-C0040-B1006-R0040 (6 colonies), b (7 colonies)
  • Gel result:
    • b (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, 12, 22, 30, 27(2nd col), b(1st col) (see "Parts" section for our nomenclature).
  • Single colonies plates for b, c, d.

08/5/08

  • Glycerol stocks/miniprep for C0062, 12, 22, 30, 27(2), b(1).
  • We sent C0062, 12, 22 and 30 to Primm for sequencing: all these parts contain BBa_C0062.
  • We transformed/plated 28.
  • Colony PCR for a (7 colonies), b(single colonies)(6 colonies), c(single colonies)(6 colonies), d(single colonies)(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:
    • a (1, 4, 6, 7)
    • b (no colony was chosen: we already had them and this run didn't show any 100% pure colony)
    • c (5)
    • d (2)

08/6/08

  • Single colonies plate for 28, because where were too many bacteria on its plate.
  • Glycerol stocks/miniprep for:
    • a(1)
    • a(4)
    • a(6)
    • a(7)
    • c(5)
    • d(2)
  • We cut these 6 plasmids E-P (5 µl of DNA in a final reaction volume of 20 µl).
  • Run for digested plasmids. Gel results:
    • a(1) OK
    • a(4) False positive
    • a(6) OK
    • a(7) False positive
    • c(5) OK
    • d(2) OK

08/7/08

  • Colony PCR for 28 single colonies plate (screening on 6 colonies).
  • Gel results: all screened colonies were negative...
Marker 1Kb, blank, J23100-B0030-C0040-B1006-R0040 (6 colonies), b (7 colonies)
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