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Team:Osaka/Notebook
From 2010.igem.org
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| - | + | |<span style={{{s1|"color:{{{c1|red}}};"}}}>1</span> | |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 2 (Mon)|2]]<span style={{{s2|"color:{{{c2|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 3 (Tue)|3]]<span style={{{s3|"color:{{{c3|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 4 (Wed)|4]]<span style={{{s4|"color:{{{c4|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 5 (Thu)|5]]<span style={{{s5|"color:{{{c5|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 6 (Fri)|6]]<span style={{{s6|"color:{{{c6|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 7 (Sat)|7]]<span style={{{s7|"color:{{{c7|deepskyblue}}};"}}}></span> |
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| - | |[[ | + | |[[Team:Osaka/Notebook#August 8 (Sun)|8]]<span style={{{s8|"color:{{{c8|red}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 9 (Mon)|9]]<span style={{{s9|"color:{{{c9|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 10 (Tue)|10]]<span style={{{s10|"color:{{{c10|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 11 (Wed)|11]]<span style={{{s11|"color:{{{c11|inherit}}};"}}}></span> |
| - | + | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> | |
| - | + | |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> | |
| - | + | |<span style={{{s14|"color:{{{c14|deepskyblue}}};"}}}>14</span> | |
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| - | + | |<span style={{{s15|"color:{{{c15|red}}};"}}}>15</span> | |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 16 (Mon)|16]]<span style={{{s16|"color:{{{c16|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 17 (Tue)|17]]<span style={{{s17|"color:{{{c17|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 18 (Wed)|18]]<span style={{{s18|"color:{{{c18|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 19 (Thu)|19]]<span style={{{s19|"color:{{{c19|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 20 (Fri)|20]]<span style={{{s20|"color:{{{c20|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 21 (Sat)|21]]<span style={{{s21|"color:{{{c21|deepskyblue}}};"}}}></span> |
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| - | |[[ | + | |[[Team:Osaka/Notebook#August 22 (Sun)|22]]<span style={{{s22|"color:{{{c22|red}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 23 (Mon)|23]]<span style={{{s23|"color:{{{c23|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 24 (Tue)|24]]<span style={{{s24|"color:{{{c24|inherit}}};"}}}></span> |
| - | |[[ | + | |[[Team:Osaka/Notebook#August 25 (Wed)|25]]<span style={{{s25|"color:{{{c25|inherit}}};"}}}></span> |
| - | + | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> | |
| - | + | |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> | |
| - | + | |<span style={{{s28|"color:{{{c28|deepskyblue}}};"}}}>28</span> | |
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| - | + | |<span style={{{s29|"color:{{{c29|red}}};"}}}>29</span> | |
| - | + | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> | |
| - | + | |<span style={{{s31|"color:{{{c31|inherit}}};"}}}>31</span> | |
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| - | + | |<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span> | |
| - | + | |<span style={{{s2|"color:{{{c2|inherit}}};"}}}>2</span> | |
| - | + | |<span style={{{s3|"color:{{{c3|inherit}}};"}}}>3</span> | |
| - | + | |<span style={{{s4|"color:{{{c4|deepskyblue}}};"}}}>4</span> | |
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| - | + | |<span style={{{s5|"color:{{{c5|red}}};"}}}>5</span> | |
| - | + | |<span style={{{s6|"color:{{{c6|inherit}}};"}}}>6</span> | |
| - | + | |<span style={{{s7|"color:{{{c7|inherit}}};"}}}>7</span> | |
| - | + | |<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span> | |
| - | + | |<span style={{{s9|"color:{{{c9|inherit}}};"}}}>9</span> | |
| - | + | |<span style={{{s10|"color:{{{c10|inherit}}};"}}}>10</span> | |
| - | + | |<span style={{{s11|"color:{{{c11|deepskyblue}}};"}}}>11</span> | |
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| - | + | |<span style={{{s12|"color:{{{c12|red}}};"}}}>12</span> | |
| - | + | |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> | |
| - | + | |<span style={{{s14|"color:{{{c14|inherit}}};"}}}>14</span> | |
| - | + | |<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span> | |
| - | + | |<span style={{{s16|"color:{{{c16|inherit}}};"}}}>16</span> | |
| - | + | |<span style={{{s17|"color:{{{c17|inherit}}};"}}}>17</span> | |
| - | + | |<span style={{{s18|"color:{{{c18|deepskyblue}}};"}}}>18</span> | |
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| - | + | |<span style={{{s19|"color:{{{c19|red}}};"}}}>19</span> | |
| - | + | |<span style={{{s20|"color:{{{c20|inherit}}};"}}}>20</span> | |
| - | + | |<span style={{{s21|"color:{{{c21|inherit}}};"}}}>21</span> | |
| - | + | |<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span> | |
| - | + | |<span style={{{s23|"color:{{{c23|inherit}}};"}}}>23</span> | |
| - | + | |<span style={{{s24|"color:{{{c24|inherit}}};"}}}>24</span> | |
| - | + | |<span style={{{s25|"color:{{{c25|deepskyblue}}};"}}}>25</span> | |
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| - | + | |<span style={{{s26|"color:{{{c26|red}}};"}}}>26</span> | |
| - | + | |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> | |
| - | + | |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> | |
| - | + | |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span> | |
| - | + | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> | |
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| - | + | |<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span> | |
| - | + | |<span style={{{s2|"color:{{{c2|deepskyblue}}};"}}}>2</span> | |
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| - | + | |<span style={{{s3|"color:{{{c3|red}}};"}}}>3</span> | |
| - | + | |<span style={{{s4|"color:{{{c4|inherit}}};"}}}>4</span> | |
| - | + | |<span style={{{s5|"color:{{{c5|inherit}}};"}}}>5</span> | |
| - | + | |<span style={{{s6|"color:{{{c6|inherit}}};"}}}>6</span> | |
| - | + | |<span style={{{s7|"color:{{{c7|inherit}}};"}}}>7</span> | |
| - | + | |<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span> | |
| - | + | |<span style={{{s9|"color:{{{c9|deepskyblue}}};"}}}>9</span> | |
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| - | + | |<span style={{{s10|"color:{{{c10|red}}};"}}}>10</span> | |
| - | + | |<span style={{{s11|"color:{{{c11|inherit}}};"}}}>11</span> | |
| - | + | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> | |
| - | + | |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> | |
| - | + | |<span style={{{s14|"color:{{{c14|inherit}}};"}}}>14</span> | |
| - | + | |<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span> | |
| - | + | |<span style={{{s16|"color:{{{c16|deepskyblue}}};"}}}>16</span> | |
|- | |- | ||
| - | + | |<span style={{{s17|"color:{{{c17|red}}};"}}}>17</span> | |
| - | + | |<span style={{{s18|"color:{{{c18|inherit}}};"}}}>18</span> | |
| - | + | |<span style={{{s19|"color:{{{c19|inherit}}};"}}}>19</span> | |
| - | + | |<span style={{{s20|"color:{{{c20|inherit}}};"}}}>20</span> | |
| - | + | |<span style={{{s21|"color:{{{c21|inherit}}};"}}}>21</span> | |
| - | + | |<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span> | |
| - | + | |<span style={{{s23|"color:{{{c23|deepskyblue}}};"}}}>23</span> | |
|- | |- | ||
| - | + | |<span style={{{s24|"color:{{{c24|red}}};"}}}>24</span> | |
| - | + | |<span style={{{s25|"color:{{{c25|inherit}}};"}}}>25</span> | |
| - | + | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> | |
| - | + | |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> | |
| - | + | |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> | |
| - | + | |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span> | |
| - | + | |<span style={{{s30|"color:{{{c30|deepskyblue}}};"}}}>30</span> | |
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|[[{{{prefix|}}}10.31{{{suffix|}}}|<span style={{{s31|"color:{{{c31|red}}};"}}}>31</span>]] | |[[{{{prefix|}}}10.31{{{suffix|}}}|<span style={{{s31|"color:{{{c31|red}}};"}}}>31</span>]] | ||
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| - | + | |<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span> | |
| - | + | |<span style={{{s2|"color:{{{c2|inherit}}};"}}}>2</span> | |
| - | + | |<span style={{{s3|"color:{{{c3|inherit}}};"}}}>3</span> | |
| - | + | |<span style={{{s4|"color:{{{c4|inherit}}};"}}}>4</span> | |
| - | + | |<span style={{{s5|"color:{{{c5|inherit}}};"}}}>5</span> | |
| - | + | |<span style={{{s6|"color:{{{c6|deepskyblue}}};"}}}>6</span> | |
|- | |- | ||
| - | + | |<span style={{{s7|"color:{{{c7|red}}};"}}}>7</span> | |
| - | + | |<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span> | |
| - | + | |<span style={{{s9|"color:{{{c9|inherit}}};"}}}>9</span> | |
| - | + | |<span style={{{s10|"color:{{{c10|inherit}}};"}}}>10</span> | |
| - | + | |<span style={{{s11|"color:{{{c11|inherit}}};"}}}>11</span> | |
| - | + | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> | |
| - | + | |<span style={{{s13|"color:{{{c13|deepskyblue}}};"}}}>13</span> | |
|- | |- | ||
| - | + | |<span style={{{s14|"color:{{{c14|red}}};"}}}>14</span> | |
| - | + | |<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span> | |
| - | + | |<span style={{{s16|"color:{{{c16|inherit}}};"}}}>16</span> | |
| - | + | |<span style={{{s17|"color:{{{c17|inherit}}};"}}}>17</span> | |
| - | + | |<span style={{{s18|"color:{{{c18|inherit}}};"}}}>18</span> | |
| - | + | |<span style={{{s19|"color:{{{c19|inherit}}};"}}}>19</span> | |
| - | + | |<span style={{{s20|"color:{{{c20|deepskyblue}}};"}}}>20</span> | |
|- | |- | ||
| - | + | |<span style={{{s21|"color:{{{c21|red}}};"}}}>21</span> | |
| - | + | |<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span> | |
| - | + | |<span style={{{s23|"color:{{{c23|inherit}}};"}}}>23</span> | |
| - | + | |<span style={{{s24|"color:{{{c24|inherit}}};"}}}>24</span> | |
| - | + | |<span style={{{s25|"color:{{{c25|inherit}}};"}}}>25</span> | |
| - | + | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> | |
| - | + | |<span style={{{s27|"color:{{{c27|deepskyblue}}};"}}}>27</span> | |
|- | |- | ||
| - | + | |<span style={{{s28|"color:{{{c28|red}}};"}}}>28</span> | |
| - | + | |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span> | |
| - | + | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> | |
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Revision as of 11:48, 6 October 2010
Calendar
| July | ||||||
| S | M | T | W | T | F | 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 | ||||||
| S | M | T | W | T | F | 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 | ||||
| September | ||||||
| S | M | T | W | T | F | 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 | ||||||
| S | M | T | W | T | F | 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 | ||||||
| S | M | T | W | T | F | 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 | ||||
Notebook
July 29 (Thu)
Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura
- Safety lecture for junior members.
- Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).
July 31 (Sat)
Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka
- Meeting
- Summer project schedule
- List of genes to clone
August 2 (Mon)
Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka
- Culture medium preparation
- LB agar plates (49 antibiotic-less plates)
- LB liquid medium (500 ml)
- Competent cells preparation - Nojima Method
- SOB medium (MgCl2 not yet added) -> stored at 4˚C
- TB buffer -> stored at 4˚C
- Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n
Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.
August 3 (Tue)
Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh
- Competent cells preparation (continued)
- Preparation of glucose solution for making SOC medium.
- Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
- (Night) Transfer from pre-culture to growth culture.
August 4 (Wed)
Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh
- OD measurements throughout the day till required OD (0.3~0.7) was obtained.
- Completion of competent cells according to protocol.
August 5 (Thu)
Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino
- Transformation of Registry parts:
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-20J | <bbpart>BBa_K118023</bbpart> | A | C. fermi endocellulase Cen A coding |
| 2-20H | <bbpart>BBa_K118022</bbpart> | A | C. fermi exocellulase Cex coding |
| 1-2M | <bbpart>BBa_B0034</bbpart> | A | RBS |
| 1-13D | <bbpart>BBa_B0010</bbpart> | A | terminator |
| 1-1D | <bbpart>BBa_R0010</bbpart> | A | promoter |
| 1-18F | <bbpart>BBa_E1010</bbpart> | K | RFP coding |
Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.
August 6 (Fri)
Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh
- Colony check
- All transformed cells produced colonies!
- Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
- Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C
August 7 (Sat)
Attendance: Nakamura, Saka, Yasumoto, Takino
- Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
- Transformation of construction plasmids
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 1-1C | <bbpart>pSB1A3</bbpart> | A | construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>) |
| 1-3A | <bbpart>pSB1C3</bbpart> | C | (" ") |
| 1-5A | <bbpart>pSB1K3</bbpart> | K | (" ") |
- Meeting
August 8 (Sun)
Attendance: Nakamura, Yasumoto
- Colony check
- All parts successfully transformed
- Transfer to LB culture medium
August 9 (Mon)
- Miniprep of 1-1C
- Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
- (WHICH ENZYMES?)
- Gel electrophoresis of digests ("cut check")
- 2-20H, 2-20J, 1-1C -> OK
- 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
- Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
- Yesterday's inoculated culture mediums contained the wrong antibiotics!
- Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock
August 10 (Tue)
- Miniprep of 1-3A, 1-5A
- Restriction digests of 1-3A, 1-5A
- Gel electrophoresis
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
- 1-3A, 1-5A -> OK; others -> not cut (again)
- 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
- all parts not cut
- 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
August 11 (Wed)
- Miniprep of last year's parts transformed on Monday
- Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
- all 4 not cut... AGAIN
- so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
- will try with different set of restriction enzymes next week
- Sent miniprepped last year's parts to ECUST team in Shanghai, China
August 16 (Mon)
- Restriction digests
- 1-13D, 1-2M, 1-1D, 1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
- 2-20J with XbaI, PstI to check/confirm XbaI activity
- Gel electrophoresis of digests
- (RESULTS?)
- Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D, 1-2M, 1-1D, 1-18F
- 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added
August 17 (Tue)
- Mini-prep of 1-13D, 1-2M, 1-1D, 1-18F parts inoculated yesterday (2 of each)
- 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
- 1-1D, 1-18F with EcoRI, SpeI
- 1-13D, 1-2M with XbaI, PstI
- (RESULTS?)
- Transformation of secretion tag parts using 25μl of competent cells each
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-22P | <bbpart>BBa_K103006</bbpart> | A | OmpA outer membrane protein + linker |
| 1-2J | <bbpart>BBa_J32015</bbpart> | A,K | PelB leader sequence |
August 18 (Wed)
iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda
August 19 (Thu)
- Transfer of 2-22P, 1-2J to solution culture
- Gel electrophoresis of digests from 'cut check' products from Tuesday
- repeat run, but each digest together with undigested plasmid DNA)
- 2% agarose gel instead of the usual 1%
- (RESULTS?)
- Gel electrophoresis of 1-1D digest only
- (RESULT?)
- Multiple restriction digests of 1-1D to check for problems at restriction sites
- tried the following: EcoRI only; SpeI only; EcoRI + SpeI
- Night: miniprep of 2-22P, 1-2J inoculated in the morning
August 20 (Fri)
- Gel electrophoresis of 1-1D and its digests
- (RESULTS?)
- 'Cut check' of parts miniprepped the night before
- both 2-22P & 1-2J cut with XbaI, PstI
- enzyme inactivation at 80℃, 20min
- (RESULTS?)
- Restriction digest of 2-20J (WHICH ENZYMES?)
- Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
- reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
- reaction at room temperature for 10min; ligase inactivation at 80℃ for 20min
- Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix
August 21 (Sat)
- Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
- we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
- 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
- ligation product designated as <1>; Chloramphenicol resistance
- same ligation mix composition as yesterday's
- Transformation of <1> with pre-incubation for 1.5hr instead of 1hr
August 22 (Sun)
- Transfer of <1> to culture solution; incubation at 30℃ (why??)
- Transformation of the following parts:
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-4A | <bbpart>BBa_J63005</bbpart> | A | yeast ADH1 promoter |
| F1 | N/A | A | beta-galactosidase from Edinburgh team |
| F2 | N/A | C | RBS + F1 |
| F3 | N/A | C | Lac promoter + RBS + F1 |
- O/N incubation at 37℃ as per normal
August 23 (Mon)
- Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
- Miniprep & 'Cut check' of <1>
- cut at EcoRI, SpeI
- gel run with DNA ladder, digested plasmid, undigested plasmid
- (RESULTS?)
- Transfer of 3 more colonies of <1> to liquid solution (to store as glycerol stock - see Tue notes)
- Transformation of the following registry parts
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-2O | <bbpart>J63003</bbpart> | A | yeast Kozak sequence |
| 3-11I | <bbpart>K105027</bbpart> | A | 'cyc100' minimal promoter |
August 24 (Tue)
- Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
- transfer to solution culture
- Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
- (RESULTS)
- Transfer of F1 to solution culture (why?)
- Preparation of glycerol stock of cell culture containing <1> (why?)
- 200ml of culture solution mixed with 100ml of 50% glycerol
- stored at -80℃
August 25 (Wed)
- Miniprep of parts in solution culture: 2-2O, 3-11I, F1
- Cut check of 3-11I & F1 with EcoRI, SpeI
- (RESULTS?)
