Team:UNIPV-Pavia/Notebook/Week10

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<br><br>
<br><br>
-
==Week 8: 07/14/08 - 07/17/08==
+
==Week 10: 07/21/08 - 07/25/08==
-
'''07/14/08'''
+
'''07/21/08'''
<br>
<br>
-
*We transformed 1 µl of the 6 ligations stored at -20°C. We plated transformed bacteria and incubated plates at 37°C overnight.
+
*Colony PCR for B0030-C0078-B1006-'''R0079''': 6 colonies from single colonies plate.
 +
 
 +
{|
 +
|[[Image:pv_colonypcr_25_single_col.jpg|thumb|300px|left|B0030-C0078-B1006-'''R0079''']]
 +
|}
 +
 
 +
*Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.
 +
 
 +
*We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-'''R0040''' and '''B0030'''-C0061 glycerol stocks to grow two overnight cultures.
 +
 
 +
*Ligation:
 +
**J23100-B0030-C0012-'''B1006''' (1:2 ratio, 40 ng of vector)
 +
**B0030-I15009-B1006-'''R0082''' (30 ng of vector)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006
 +
 
 +
*We incubated ligations at 16°C overnight.
 +
 
 +
<br><br>
 +
'''07/22/08'''
 +
<br>
 +
*Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.
 +
 
 +
*Glycerol stocks/miniprep for:
 +
{|cellpadding="20px"
 +
|J23100-B0030-C0040-B1006-'''R0040'''
 +
|'''B0030'''-C0061
 +
|-
 +
|B0030-C0078-B1006-'''R0079'''
 +
|}
 +
 
 +
*We sent J23100-B0030-C0040-B1006-'''R0040''' and B0030-C0078-B1006-'''R0079''' purified plasmids to Primm for sequencing.
*Plasmid digestion for:
*Plasmid digestion for:
{|cellpadding="20px"
{|cellpadding="20px"
-
|BBa_R0079 (E-X)
+
|'''B0030'''-C0061 (E-S)
-
|BBa_B0030-BBa_C0078-'''BBa_B1006''' (E-S)
+
|B0030-C0078-B1006-'''R0079''' (E-X)
|}
|}
-
*Gel run/cut/gel extraction for the 2 parts.
+
*Gel run/cut/gel extraction.
-
*Ligation: BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079'''.
+
<br><br>
 +
'''07/23/08'''
 +
<br>
 +
*Ligation plates showed colonies!We put ligation plates at +4°C.
 +
 
 +
*Ligation for B0030-C0061-B0030-C0078-B1006-'''R0079'''.
*We incubated ligation at 16°C overnight.
*We incubated ligation at 16°C overnight.
<br><br>
<br><br>
-
'''07/15/08'''
+
'''07/24/08'''
<br>
<br>
-
*We transformed 1 µl of BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' ligation and plated transformed bacteria. We incubated the plate at 37°C overnight.
+
*Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-'''R0079''' ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
-
*All the 6 overnight plates showed colonies. We performed colony PCR for (we decided to pick up more colonies for the ligations with long inserts):
+
*Colony PCR for:
-
#BBa_J23100-'''BBa_B0030''' (S-P) -BBa_C0012 (standard ligation) - 10 colonies
+
**J23100-B0030-C0012-'''B1006'''
-
#'''pSB1AK3'''-BBa_J23100-BBa_B0030-BBa_C0012 (double ligation) - 7 colonies
+
**B0030-I15009-B1006-'''R0082'''
-
#BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040''' - 3 colonies
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062
-
#BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062''' - 3 colonies
+
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006
-
#BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006''' - 10 colonies
+
 
-
#BBa_B0030-BBa_I15009-'''BBa_B1006''' - 3 colonies
+
*(We performed one PCR for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
 +
 
 +
{|cellpadding="1px"
 +
|[[Image:pv_pcr_parallel1.jpg|thumb|300px|left|Thermal cycler working on J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' colonies: 2 min elongation]]
 +
|[[Image:pv_pcr_parallel2.jpg|thumb|300px|left|Thermal cycler working on B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 colonies: 3 min elongation]]
 +
|}
 +
 
 +
*Electrophoresis (1.2% agarose) for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''':
{|
{|
-
|[[Image:pv_colonypcr_15_7_08.jpg|thumb|300px|left|Picked up colonies in LB + Amp]]
+
|[[Image:pv_colonypcr_19_32.jpg|thumb|300px|left|Marker 1Kb, blank, J23100-B0030-C0012-'''B1006''' (7 lanes), B0030-I15009-B1006-'''R0082''' (6 lanes)]]
|}
|}
 +
 +
*Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006:
 +
 +
{|
 +
|[[Image:pv_colonypcr_34_35.jpg|thumb|300px|left|Marker 1Kb, B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (7 lanes), B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (7 lanes)]]
 +
|}
 +
 +
*Gel results:
 +
**J23100-B0030-C0012-'''B1006''' 1st colony
 +
**B0030-I15009-B1006-'''R0082''' 1st colony
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 1st and 4th colony
 +
 +
*We decided to keep two colonies for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
 +
 +
*The fifth colony of B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.
 +
 +
*We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.
 +
 +
<br><br>
 +
'''07/25/08'''
 +
<br>
 +
*Glycerol stocks/miniprep for:
 +
**J23100-B0030-C0012-'''B1006''' (1)
 +
**B0030-I15009-B1006-'''R0082''' (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)
 +
 +
*We sent these purified plasmids:
 +
**B0030-I15009-B1006-'''R0082''' (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1)
 +
**B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4)
 +
 +
*to Primm for sequencing.
 +
 +
*We put B0030-C0061-B0030-C0078-B1006-'''R0079''' plate at +4°C.

Latest revision as of 21:26, 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 10: 07/21/08 - 07/25/08

07/21/08

  • Colony PCR for B0030-C0078-B1006-R0079: 6 colonies from single colonies plate.
B0030-C0078-B1006-R0079
  • Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.
  • We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-R0040 and B0030-C0061 glycerol stocks to grow two overnight cultures.
  • Ligation:
    • J23100-B0030-C0012-B1006 (1:2 ratio, 40 ng of vector)
    • B0030-I15009-B1006-R0082 (30 ng of vector)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
  • We incubated ligations at 16°C overnight.



07/22/08

  • Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.
  • Glycerol stocks/miniprep for:
J23100-B0030-C0040-B1006-R0040 B0030-C0061
B0030-C0078-B1006-R0079
  • We sent J23100-B0030-C0040-B1006-R0040 and B0030-C0078-B1006-R0079 purified plasmids to Primm for sequencing.
  • Plasmid digestion for:
B0030-C0061 (E-S) B0030-C0078-B1006-R0079 (E-X)
  • Gel run/cut/gel extraction.



07/23/08

  • Ligation plates showed colonies!We put ligation plates at +4°C.
  • Ligation for B0030-C0061-B0030-C0078-B1006-R0079.
  • We incubated ligation at 16°C overnight.



07/24/08

  • Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
  • Colony PCR for:
    • J23100-B0030-C0012-B1006
    • B0030-I15009-B1006-R0082
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
  • (We performed one PCR for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
Thermal cycler working on J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 colonies: 2 min elongation
Thermal cycler working on B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 colonies: 3 min elongation
  • Electrophoresis (1.2% agarose) for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082:
Marker 1Kb, blank, J23100-B0030-C0012-B1006 (7 lanes), B0030-I15009-B1006-R0082 (6 lanes)
  • Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006:
Marker 1Kb, B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (7 lanes), B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (7 lanes)
  • Gel results:
    • J23100-B0030-C0012-B1006 1st colony
    • B0030-I15009-B1006-R0082 1st colony
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 1st and 4th colony
  • We decided to keep two colonies for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
  • The fifth colony of B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.
  • We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.



07/25/08

  • Glycerol stocks/miniprep for:
    • J23100-B0030-C0012-B1006 (1)
    • B0030-I15009-B1006-R0082 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
  • We sent these purified plasmids:
    • B0030-I15009-B1006-R0082 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
    • B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
  • to Primm for sequencing.
  • We put B0030-C0061-B0030-C0078-B1006-R0079 plate at +4°C.
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