IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week9/Chemical and Light: Difference between revisions
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Lane 4: P115 cut ES (960; 2750) | Lane 4: P115 cut ES (960; 2750) | ||
Lane 5: 1 kb ladder | Lane 5: 1 kb PLUS ladder | ||
Revision as of 11:06, 19 August 2008
105, mtrB Ligations/transformations 08/17
Parts to ligated came from 8/17
We used a 2:1 molar ratio of insert to vector for all of the ligations. The ligations were all transformed into TOP10 cells.
| Plasmid | Number of colonies |
| P105+P1'BB | 2 |
| P105+P3'BB | 102 |
| P101+P105 | 0 |
| P101+P108 | 0 |
| (mtrB(BB)+P97)+P108 | 20 |
| (mtrB(BB)+P97)+P116 | 4 |
| (mtrB(BB)+P97)+P117 | 13 |
In retrospect, it appears that all the ligations with mtrB are wrong, since the digest was actually wrong.
105 Colony PCR 08/18
The annealing temperature was 55 °C and the elongation time was 2'45" (we used Platinum Taq supermix).
Expected band size is 2475bp.
Gel 1: 1-11
- #2, 5 picked for 5mL culture
Lane 1: 1 kb ladder
Lanes 2-3: P105+P1'BB
Lanes 4-12: P105+P3'BB
Gel 2: 12-22
- #12, 14 picked for 5mL culture
Lane 1: 1 kb ladder
Lanes 2-12: P105+P3'BB
RE digests 45, 63, 97 08/18
We used the Fermentas enzymes and digested for 25'.
Lane 1: 1 kb ladder
Lane 2: P45 cut XP (876; 2079)
Lane 3: P63 cut EX (3284)
Lane 4: P97 cut SP (2091)
QPI+45 Ligations/transformations 08/18
We used the 45 just digested, so all DNA was cut with the Fermentas enzymes.
| Plasmid | Ratio (insert!vector) | Strain | Number of colonies |
| P117? vector control | 2!1 molar with EB | DH5-alpha | 1 |
| P117?+45 | 2!1 molar | TOP10 | 2 |
| P117?+45 | 2!1 molar | DH5-alpha | 30 |
| P116 vector control | 2!1 molar with EB | DH5-alpha | 0 |
| P116+45 | 2!1 molar | TOP10 | 5 |
| P116+45 | 2!1 molar | DH5-alpha | 50 |
| P108 vector control | 2!1 molar with EB | DH5-alpha | 2 |
| P108+45 | 2!1 molar | DH5-alpha | 4 |
| P108+45 | 2!1 molar | TOP10 | 1 |
| P108+45 | 7!1 volumetric | DH5-alpha | 0 |
Colony PCR 08/19
The annealing temperature was 55°C and the elongation time was 2'30" for P108+45 and 2' for P116 and P117. We used the Platinum Taq SUPERmix.
Gel 1
Lane 1: 1 kb plus ladder
Lanes 2-5: P108+45
Lanes 6-12: P116+45
Gel 2
Lane 1: 1 kb plus ladder
Lanes 2-12: P116+45
Gel 3
Lane 1: 1 kb plus ladder
Lanes 2-3: P116+45
Lanes 4-12: P117+45
RE digests 101, 115, 118 08/19
Lane 1: P101 cut ES (3221; 1090)
Lane 2: P101 cut XP (3221; 1090)
Lane 3: P118 cut XP (1555; 2750)
Lane 4: P115 cut ES (960; 2750)
Lane 5: 1 kb PLUS ladder
