IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week9/Chemical and Light: Difference between revisions
Meng Xiao He (talk | contribs) |
|||
Line 90: | Line 90: | ||
|} | |} | ||
=Colony PCR 08/18= | ===Colony PCR 08/18=== | ||
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. | 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. | ||
Revision as of 09:19, 19 August 2008
105, mtrB Ligations/transformations results 8/18
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 08/18
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