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.

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 (~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 08/20

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)