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

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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

8-18 colpcr ta1.jpg

  • #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

8-18 colpcr ta2.jpg

  • #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'.

8-18 gel 1 MXHTA.jpg

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

8-19 PCR 1 MXHTA.jpg

Lane 1: 1 kb plus ladder

Lanes 2-5: P108+45 (~2200)

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

Gel 2

8-19 PCR 2 MXHTA.jpg

Lane 1: 1 kb plus ladder

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

Gel 3

8-19 PCR 3 MXHTA.jpg

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

8-19 gel 1 MXHTA.jpg

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).

Plasmid Strain Number of colonies
P101 vector control (P115) DH5-alpha too many to count (same as P101+115 in DH5-alpha)
P101+115 DH5-alpha too many to count
P101+115 TOP10 too many to count (more than in P101+115 in DH5-alpha)
P101 vector control (P115) DH5-alpha too many to count (less than P101+118 in DH5-alpha)
P101+115 DH5-alpha too many to count
P101+115 TOP10 too many to count (more than in P101+118 in DH5-alpha)

Colony PCR 08/20

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

8-20 Colony PCR 2 MXHTA .png

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.

8-20 mtrB PCR MXHTA.jpg

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


8-21 mtrBrbs.jpg

All conditions failed.

RE digests 08/20

8-20 gel 1 MXHTA.jpg

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)