IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week9/Chemical and Light

=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.

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
Lane 1: 1 kb ladder
 * #2, 5 picked for 5mL culture

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.

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 (~2200)

Lanes 6-12: P116+45 (~1800)

Gel 2



Lane 1: 1 kb plus ladder

Lanes 2-12: P116+45 (~1800)

Gel 3



Lane 1: 1 kb plus ladder

Lanes 2-3: P116+45 (~1800)

Lanes 4-12: P117+45 (~1800)

=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

Ligations/transformations of P101+115/118 08/19
We used a 2:1 insert to vector ratio for all ligations. We tried using Amy™'s method of dephosphorylating (i.e. using the Roche kit).

Colony PCR 08/20
We used an annealing temperature of 55 °C and elongation temperature 1'45" and 2'15", and Platinum Taq SUPERmix



Lanes 1-2: P101 (115) vector control

Lanes 3-48: P101+115

Lanes 49-50: P101 (118) vector control

Lanes 51-96: P101+118


 * Samples 2, 4, 14, 25, 49, 61, 77 picked for 5mL culture and sequencing.


 * It's a bit alarming that the vector controls have ~1kb inserts. Uncut P101 should not transform, as the part contained is lethal (ccdB).

=mtrB+RBS PCR 8/20=

Rx mix (split into 12):
 * 120μL 5x HF buffer
 * 12μL 10mM dNTPs
 * 15μL each primer
 * 6μL resuspended wt S. oneidensis colony
 * 6μL Phusion polymerase
 * 426μL H2O

Rx conditions
 * Initial denaturation: 2:30s @ 98°C
 * Denaturations: 10s @ 98°C
 * Annealing: 30s @ 55-62°C gradient
 * Extension: 45s for first 10 cycles, 55s for next 30 cycles @ 72°C

All the tubes were mysteriously pink after thermocycling.



Attempt 2
Phusion

Rx mix (split into 12):
 * 120μL 5x HF buffer
 * 12μL 10mM dNTPs
 * 15μL each primer
 * swirl of wt S. oneidensis colony
 * 6μL Phusion polymerase
 * 432μL H2O

Rx conditions
 * Initial denaturation: 2:30s @ 98°C
 * Denaturations: 30s @ 98°C for first 10 cycles, 10s for next 35 cycles
 * Annealing: 30s @ 58-64°C gradient
 * Extension: 45s for first 10 cycles, 55s for next 35 cycles @ 72°C

Platinum

Rx mix (split into 2):
 * 135μL Supermix
 * 3μL each primer
 * swirl of wt S. oneidensis colony
 * 9μL H2O

Rx conditions
 * Initial denaturation: 10m @ 95°C
 * Denaturations: 45s @ 95°C for first 10 cycles, 30s for next 30 cycles
 * Annealing: 30s @ 57°C
 * Extension: 2:30s @ 72°C



All conditions failed.

=RE digests 08/20=

Lane 1: 1 kb plus ladder

Lane 2: P48 uncut (3477)

Lane 3: P48 cut X (3477)

Lane 4: P113 cut XP (534; 3339)

Lane 5: P117 cut SP (3687)

Lane 6: P116 cut SP (3687)

Lane 7: P38+51 (in p15A) B uncut (4155)

Lane 8: P38+51 (in p15A) B cut EP (1405)

Lane 9: P38+51 (in p15A) C uncut (4155)

Lane 10: P38+51 (in p15A) C cut EP (1405)

Lane 11: P38+51 (in p15A) D uncut (4155)

Lane 12: P38+51 (in p15A) D cut EP (1405)