IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week11/Chemical and Light

Retransformations 9/15

P126 and 127 retransformed into DH5α. Split 30μL and rest onto 2 diff CM plates.

9/16: Retransform went fine, although still fewer cells/DNA could have been used. Colonies picked for overnight cultures.

Ligations 9/15

• Tried the Takara ligation kit: 1μL vector, 4μL insert, 5μL ligation mix
• Transformed into DH5α

mtrB

P63 EX (8/14, dephos) with:

• mtrB TOPO ES (8/15): 0 colonies (on KAN)
• mtrB BB ES (8/14): 3 colonies (on KAN)

P97 SP (dephos) with:

• mtrB BB XP (8/14): TMTC (on AMP)
• mtrB TOPO XP (8/15): TMTC (on AMP)

It seems like there's something wrong with the P97, so if the colony PCRs indicate there's nothing in the plasmids, the tube'll be tossed.

Colony PCR

The 3 mtrB+63 colonies and 4 of each mtrB+97 colonies were picked for colony PCR:

Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H₂O

Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, 2m40s at 72°C), 7min final extension, hold at 4°C

2KB ladder used. Clearly, none of the clones actually had a 2kb+ band, so mtrB was not incorporated.

Lac QPI

P3+51 EX (8/3, dephos) with P38 oligo mix (Amy's prep)

0 colonies (on KAN)

RE digests 9/17: mtrB TOPO, 101, 102, 116, 117

The following digests were performed using the Fermentas protocol for 25 min: mtrB TOPO ES; mtrB TOPO XP; P63 EX; P97 SP, P101, 102, 116, 117 ES

Aside from 116, 117, the bands were extracted using a clonewell gel. 116 and 117 were not visible.

P63, P97, and P101 were dephosphorylated according to the NEB instructions using Antarctic phosphatase, and the phosphatase was heat killed.

Ligations 9/18

The following ligations were performed (using 1:6 vector:insert ratio):

• mtrB TOPO ES + P63 EX
• mtrB TOPO XP + P97 SP
• P101 ES + P102 ES

The DNA was mixed, heated to 65°C for 2 minutes, and then mixed with ligase.

• With Takara kit, 6μL DNA mix was added to 6μL ligation mix
• With NEB kit, 9μL DNA mix was added to 1.1μL T4 ligation buffer and 1μL T4 ligase

The mix was cooled to 16°C for 45min and then held at 4°C prior to transformation.

mtrB PCR

Tried PCR again with primers that incorporate RBS and Platinum Pfx.

w/ cells	'	'	'	w/ TOPO plasmid	'	'
		12x				12x
Amp buffer	5	60		Amp buffer	5	60
10mM dNTP	1.5	18		10mM dNTP	1.5	18
50 mM MgSO4	1	12		50 mM MgSO4	1	12
Primer F	0.75	9		Primer F	0.75	9
Primer R	0.75	9		Primer R	0.75	9
Pfx	0.75	9		Pfx	0.75	9
Water	40.25	483		Water	40	480
				DNA	0.25	3
w/PCR enhancer
Amp buffer	5	60		Amp buffer	5	60
10mM dNTP	1.5	18		10mM dNTP	1.5	18
50 mM MgSO4	1	12		50 mM MgSO4	1	12
Primer F	0.75	9		Primer F	0.75	9
Primer R	0.75	9		Primer R	0.75	9
Pfx	0.75	9		Pfx	0.75	9
PCRx	5	60		Water	40	480
Water	35.25	423		PCRx	5	60
				DNA	0.25	3