From 2008.igem.org

PCR

8/4: Cph/EnvZ, P1, P3, mtrB not BB

	1% agarose gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-5	cph-EnvZ 50 °C annealing temperature (2.3kb)
	6-7	P1 (3kb)
	8-9	P3 (3kb)
	10-11	mtrB (2.1kb)
	12-13	HO-pcyA (1.4kb)
	14-15	ompR (750bp)

HO-pcyA and ompR bands excised, purified, and digested (ApaLI & Kpn overnight).

8/4 Colony PCR of putative mtrB samples

We picked 24 colonies each of old putative P98 and newly transformed putative P98+63 for colony PCR using the BioBrick primers and 55°C annealing for 30 cycles. If the colonies contained mtrB, a band at ~2.1 kb should appear. No colonies appeared to contain mtrB.

All gels with 1kb plus ladder

P98	P98	P98+63

P98+63	Mix
		Lane 1: 1 kb plus ladder Lane 2: P98+63 (~2100) Lane 3: P98+63 (~2100) Lane 4: P98 (~2100) Lane 5: P98 (~2100)

8/4 CphEnvZ, P1, P3, mtrB

Pl05 (CphEnvZ w/ PstI site mutated out) conditions: 5min @ 94°C → 40x [30s @ 94°C → 30s @ (50.8, 51.5, 52.6, 53.9, 55.4, 56.7, 57.7, 58.3)°C → 2:30 @ 72°C] → 7min @ 72°C → ∞ @ 4°C

8/5: gels

	1.2% agarose E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1-8	P105 (50.8-58.3 annealing temp, increases LTR) - 2.2kb
	9	1kb plus ladder
	10	P1 not BB - 3kb
	11	P3 not BB - 3kb
	12	mtrB not BB - 2.1kb

	Gel for excision (includes old PCR samples)
	Lane	Contents (approx. expected band size)
	1, 15	1kb plus ladder
	2-3	P105 PCR product
	4-6	P3 PCR product
	7-9	mtrB not BB PCR product
	10	P1 PCR product
	11-12	ompR PCR product (720bp)
	13	HO-pcyA (1.4kb)
	14	CDF PCR product (900bp)

Bands excised and digested: Cph/P105 (AK), P3 (AK), mtrB (XP, ES)

Bands excised and saved: ompR, HO-pcyA, CDF

Ligations and transformations 08/06

We ligated P105 and P1/P3 not BB, and mtrB not BB and P1/P3 not BB. We tried letting the ligation reactions run for 5 and 10 minutes. We used P1/P3 not BB from 08/05 and from another date (older). We transformed using TOP10 and DH5α. We also retransformed P108, the Lac QPI with a high constitutive promoter in a p15A vector.

Results below

TOPO cloning

08/04

P1, mtrB

These had a few white colonies. Colony PCR, however, indicated that the correct PCR product was not incorporated.

ompR

All colonies were blue.

HO-pcyA

No colonies.

Transforming in XL10-Gold cells

Plate	Marker	# Colonies	Description
XL10-Gold P75a + P63b (1:1)	Kan	2	No fluor
XL10-Gold P75b + P63b (1:1)	Kan	6	1 fluor
XL10-Gold P75a + P63b (6:2)	Kan	0
XL10-Gold P75b + P63b (6:2)	Kan	1	fluor
XL10-Gold P75a + P63b (7:1)	Kan	1	no fluor
XL10-Gold P75b + P63b (7:1)	Kan	2	both fluor
XL10-Gold Dephos P75a	Kan	1
XL10-Gold Dephos p75b	Kan	0
XL10-Gold P3 + (P39+p51) 2/6	Kan
XL10-Gold P3 Topo w/ XGAL	Amp
XL10-Gold P1 Topo w/ XGAL	Amp
XL10-Gold mtrB Topo w/ XGAL	Amp
XL10-Gold pUC18	Amp	100s	White, very small
XL10-Gold negative control	Amp	many	extremely small
XL10-Gold negative control	Kan	0

Ligations

HO-pcyA, ompR, CDF

All three ligations yielded >50 colonies on the TOP10 cell plate with a 5 minute ligation in all combinations (HO-pcyA and ompR with P1 and old P3; CDF with P3). The P90 (CDF+P3) did not fluoresce. The DH5α cells did not have nearly as many (<5) and ligation times (10min, 30 min) did not appear to be significant.

Colony PCR 8-6

We picked at least 18 colonies from each ligation set, patched a master plate, inoculated liquid cultures, and performed 12.5 μL colony PCR reactions. The BBp/sfx primer pair was used, annealing temperature was 50°C for first 10 cycles and 54°C for rest of the cycles. 2 controls were performed: one using a picked P20 colony, and another with miniprepped P20 plasmid.

Digests, 1-8

We ran the digests to see if PCR mutations could have introduced an internal cut site. However, even with the small amount of purified digest we had left, we could see a properly sized band for each of P1, ompR, and HO-pcyA PCR products.

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2	Old P1 PCR product AK digested (3kb)
	3	ompR PCR product AK digested (720bp)
	4	HO-pcyA PCR product AK digested (1.4kb)
	5-12	HO-pcyA + P1 ligation colony PCR (1.6kb)

9-19

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-11	HO-pcyA + P1 ligation colony PCR (1.6kb)
	12	HO-pcyA + P3 ligation colony PCR (1.6kb)

20-30

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-12	HO-pcyA + P3 ligation colony PCR (1.6kb)

31-41

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-7	HO-pcyA + P3 ligation colony PCR (1.6kb)
	8-12	CDF + P3 ligation colony PCR (900bp)

42-52

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-12	CDF + P3 ligation colony PCR (900bp)

53-63

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	1 kb plus ladder
	2-7	CDF + P3 ligation colony PCR (900bp)
	8-12	ompR + P1 ligation colony PCR (1kb)

64-74

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1-11	ompR + P1 ligation colony PCR (1kb) (#64 and 73 contain ompR, as verified by sequencing)
	12	1 kb plus ladder

75-85

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1-2	ompR + P1 ligation colony PCR (1kb)
	3-11	ompR + P3 ligation colony PCR (1kb)
	12	1 kb plus ladder

86-94, P20 controls

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1-9	ompR + P3 ligation colony PCR (1kb)
	10	P20 colony PCR (1122)
	11	P20 plasmid PCR
	12	1 kb plus ladder

Making Thermoinducible cI System

Ligation from 8/1

Plate Results (also listed in Week 6):

Plate	Marker	# Colonies	Description
E1 P75a + P63b (1:1)	Kan	0
E1 P75b + P63b (1:1)	Kan	0
E1 P75a + P63b (6:2)	Kan	1	No Fluor, 3mm
E1 P75b + P63b (6:2)	Kan	1	Fluorescent, 1mm.
E1 P75a + P63b (7:1)	Kan	0
E1 P75b + P63b (7:1)	Kan	0
Dephos P75b	Kan	0

Fluorescent colony was picked for liq culture and restreaked. Miniprep sent for sequencing-- ligation confirmed by sequencing (8/5).

Analytical Digest Gels from TOPO Cloning 8/5

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	E1 RBS 4R, 2D 1
	3	E1 RBS 4R, 2D 2
	4	E1 RBS 4R, 2D 3
	5	E1 RBS 4R, 2D 4
	6	E1 BIG 4R, 2D 1
	7	E1 BIG 4R, 2D 2
	8	E1 BIG 4R, 2D 3
	9	E1 BIG 4R, 2D 4
	10	E1 RBS 2R, 2D Xg 1
	11	E1 RBS 2R, 2D Xg 2
	12	E1 RBS 2R, 2D Xg 3

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	BIG 4R, 4D Xg 1
	2	BIG 4R, 4D Xg 2
	3	BIG 4R, 4D Xg 3
	4	mtrB 0.5 A
	5	mtrB 0.5 B
	6	mtrB 2 A
	7	mtrB 2 B
	8	RBS gel pur 2 A
	9	RBS gel pur 2 B
	10	BIG gel pur 2 A
	11	BIG gel pur 2 B
	12	1kB Ladder

Strange bands, but sent several clones for sequencing anyway.

Digestion and Ligation of P18+P63 and P75+P63

Done in tandem and side by side with Christina (except for digestion).

Digestion of P18, P63, and P75 8/4

'	P63	P75b	P18
DNA	30 uL	6 uL	11 uL
100x BSA	0.5 uL	0.25 uL	0.25 uL
10x Buffer	5 uL Buffer 2	2.5 uL Buffer 2	2.5 uL Buffer 2
RE 1	EcoRI 1 uL	EcoRI 1 uL	EcoRI 1 uL
RE 2	SpeI 1 uL	XbaI 1 uL	XbaI 1 uL
Water	12.5 uL	14.25 uL	9.25 uL
Volume	50 uL	25 uL	25 uL

Gel of Digestion 8/5

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	Christina\'s Vector (CE)
	3	Christina\'s Insert (CI)
	4	Christina\'s Vector (IE)
	5	Christina\'s Insert (IE)
	6	P75 EX
	7	P63 ES
	8	P18 EX

Ligation of P18+P63 and P75+P63 8/5

Used dephosphorylated and undephosphorylated vector.

Plate Results 8/6

Plate	# Colonies	Notes
pUC19 Amp I	31
Negative controls:	.	.
AmpI just cells	0
AmpI ligC dephos. IE vector only	8
AmpI ligC Ephos IE vector only v+i	1
AmpI ligC G8 SP just vector	6
AmpC ligC G8 SP just vector	7
AmpC ligC dephos IE vector only	5
DNA Positive controls:	.
AmpC ligC G8 + G10	>200
AmpI ligC G8 + G10	81
AmpI ligI G8 + G10	78
AmpC ligI G8 + G10	127
whole thing:	.
AmpI ligC CE CG	103
AmpI ligC CE dephos CG	45
AmpC ligC CE dephos	57
AmpC ligC CE CG	224
uh oh:	.
AmpI ligC IE CG	3
AmpC ligC IE CG	7
AmpC ligC IE dephos V + I	46
iGEM DNA	.
AmpI (-)	3
AmpI (- Dephos)	0
Kan 63+75	64
Kan 63 + 75 Dephos	0
AmpI 63+ 18	100+
AmpI 63+18 dephos	40

Digestion and Ligation of P63 + cI857 (Both Primer Sets)

Digestion of cI857 PCR product and P63 8/5

'	P63 EX	cI857 RBS (GP) ES	cI857 BIG (GP) ES	cI857 RBS (PP) ES	cI857 BIG (PP) ES	P104 EX
DNA	10 uL	3 uL	7 uL	10 uL	10 uL	10 uL
100X BSA	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL
10X Buffer 2	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL
EcoRI	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL
Enzyme 2	1 uL XbaI	1 uL SpeI	1 uL SpeI	1 uL SpeI	1 uL SpeI	1 uL XbaI
Water	10.25 uL	17.25 uL	13.25 uL	10.25 uL	10.25 uL	10.25 uL
Volume	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL

Christina digested the same samples of P63, cI857 RBS GP and PP, and cI857 BIG GP and PP with EcoRI buffer. P104 was cut 8/6 in the morning.

Gel results 8/6

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	P63
	3	P63c
	4	P104
	5	1 kB Ladder

Ligation of P63, P63c, and P104 with cI857 8/6

Accidentally mixed P63c and P104 so plated each of those ligations in Amp and Kan selective plates.

Plate results 8/7

Plate	Final Insert Size	Marker	# Colonies
E1 P63 + RBS PP	770	Amp	28 big
E1 P63 + RBS GPC	770	Amp	25 big, 25 small
E1 P63 + BIG PP	1695	Amp	50 big, 50 small
E1 P63 + BIG GPC	1695	Amp	25 big, 25 small
E1 P63c + RBS PP	770	Amp	50 big, 50 small
E1 P63c + RBS GPC	770	Amp	25 big, 25 small
E1 P63c +BIG PP	1695	Amp	25 big, 50 small
E1 P63c + BIG GPC	1695	Amp	10 big, 25 small
E1 P63 + RBS PP 6:2	770	Amp	~50
E1 P63 + RBS PP 4:4	770	Amp	11
E1 P63 + BIG PP (No dephos)	1695 or 1600	Amp	0
E1 P63 Dephos only	N/a	Amp	0
E1 P63c Dephos only	N/a	Amp	10 big, 25 small
E1 P104 No dephos	1020	Amp	0
E1 P104 Dephos only	n/a	Amp	75-100
E1 P104 + RBS GP	1695	Amp	50
E1 P104 + RBS PP	1695	Amp	50
E1 P104 + BIG GP	2620	Amp	0
E1 P104 + BIG PP	2620	Amp	0
E1 P104 + RBS GPC	1695	Amp	3
E1 P104 + RBS PPC	1695	Amp	5
E1 P104 + BIG GPC	2620	Amp	0
E1 P104 + BIG PPC	2620	Amp	4
puc 19 (+) ctrl	N/a	Amp	50-75
(-) ctrl just cells	N/a	Kan	0
E1 P63c + RBS PP	770	Kan	~20
E1 P63c + RBS GPC	770	Kan	3 big (2mm)
E1 P63c +BIG PP	1695	Kan	0
E1 P63c + BIG GPC	1695	Kan	0
E1 P63c Dephos only	N/a	Kan	0
E1 P104 No dephos	1020	Kan	0
E1 P104 Dephos only	n/a	Kan	0
E1 P104 + RBS GP	1695	Kan	0
E1 P104 + RBS PP	1695	Kan	16 big (2mm)
E1 P104 + BIG GP	2620	Kan	0
E1 P104 + BIG PP	2620	Kan	0
E1 P104 + RBS GPC	1695	Kan	1
E1 P104 + RBS PPC	1695	Kan	0
E1 P104 + BIG GPC	2620	Kan	0
E1 P104 + BIG PPC	2620	Kan	0

Colony PCRs of 8/6 Ligations 8/7

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB ladder
	2	E1 P63 + RBS PP A
	3	E1 P63 + RBS PP B
	4	E1 P63 + RBS PP 4:4 A
	5	E1 P63 + RBS PP 4:4 B
	6	E1 P63 + RBS PP 4:4 B (well leaked)
	7	E1 P63 + RBS PP 6:2 A
	8	E1 P63 + RBS PP 6:2 B
	9	E1 P63 + RBS GPC A
	10	E1 P63 + RBS GPC B
	11	E1 P63c + RBS PP A
	12	E1 P63c + RBS PP B
	13	E1 P63 + RBS GPC A

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	E1 P63 + RBS GPC B
	2	E1 P104 + RBS PP A
	3	E1 P104 + RBS PP B
	4	E1 P104 + RBS GP A
	5	E1 P104 + RBS GP B
	6	E1 P104 + RBS PPC A
	7	E1 P104 + RBS PPC B
	8	E1 P104 + BIG GPC B
	9	E1 P104 + BIG GPC A
	10	E1 P104 Dephos only A
	11	1 kB Ladder
	12	E1 P104 Dephos only B
	13	E1 P63c Dephos only A
	14	E1 P63c Dephos only B
	15	E1 P63 + BIG PP A
	16	E1 P63 + BIG PP B
	17	E1 P63 + BIG GPC A
	18	E1 P63 + BIG GPC B
	19	E1 P63c +BIG PP A
	20	E1 P63c +BIG PP B

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	E1 P63c + BIG GPC A
	2	E1 P63c + BIG GPC B
	3	E1 P104 + BIG PPC A
	4	E1 P104 + BIG PPC B
	5	E1 P104 + RBS GPC A
	6	E1 P63c + RBS GPC A
	7	E1 P63c + RBS GPC B
	8	E1 P104 + RBS PP A
	9	E1 P104 + RBS PP B
	10	E1 P104 + RBS PP C
	11	E1 P104 + RBS PP D
	12	E1 P104 + RBS PP E
	13	E1 P63c + RBS PP A
	14	E1 P63c + RBS PP B
	15	E1 P63c + RBS PP C
	16	E1 P63c + RBS PP D
	17	E1 P63c + RBS PP E
	18	E1 P63c + RBS PP F
	19	E1 P63c + RBS PP G
	20	1 kB Ladder

E1 P63 + RBS PP B to be sequenced Monday. All of the bands in the third gel indicated re-ligated vector.

Ligation of P63, P63c, and P104 with cI (repeat) 8/7

Ligations were re-done with newly digested vector because of mishap in 8/6 ligations.

Digestions of P63, P63c, and P104 8/6

'	P63 EX	P63c EX	P104 EX
DNA	10 uL	10 uL	10 uL
100X BSA	0.25 uL	0.25 uL	0.25 uL
10X Buffer	2.5 uL of Buffer 2	2.5 uL of EcoRI Buffer	2.5 uL of Buffer 2
EcoRI	1 uL	1 uL	1 uL
Enzyme 2	1 uL XbaI	1 uL XbaI	1 uL XbaI
Water	10.25 uL	10.25 uL	10.25 uL
Volume	25 uL	25 uL	25 uL

Gel of Digestion 8/7

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	P63
	3	P63c
	4	P104
	5	1 kB Ladder

Bands were extracted, purified, and dephosphorylated.

Ligation of P63, P63c, and P104 with cI 857 8/7

Vector backbones were ligated with cI857 digested 8/5.

Plate Results 8/8

Plate	Final Insert Size	Marker	# Colonies (After 1 Day)	# Colonies After 1 Day
E1 P63 (Buffer 2) + RBS GP		Amp	0	N/a
E1 P63 (Buffer 2) + BIG PPC		Amp	5 1-1.5mm colonies	N/a
E1 P63c + RBS GP		Amp	1 1.5mm colony	N/a
E1 P63c +BIG PPC		Amp	6 1-1.5mm colonies	N/a
E1 P63 (2) Dephos only		Amp	2 1mm colonies on edge	N/a
E1 P63c Dephos only		Amp	1 1.5mm colony	N/a
E1 P104 + RBS GP 6:2		Kan	3 colonies, no fluor	3 2-3mm colonies, 6 1mm colonies
E1 P104 + RBS GP		Kan	8 colonies, no fluor	8 2-3mm colonies, 3 1mm colonies
E1 P104 + RBS PP		Kan	30 colonies, 10 fluor, rest no fluor	30 big 2-3mm colonies, 2 1mm colonies.
E1 P104 + BIG GP		Kan	4 small 0.25 colonies?	4 1mm colonies.
E1 P104 + BIG PP		Kan	0 colonies	5 small 0.25mm colonies.
E1 P104 + RBS GPC		Kan	3 1.5mm colonies, no fluor	3 big 3mm colonies, 2 1mm colonies.
E1 P104 + RBS PPC		Kan	1 0.5mm colony	1 2mm colony, no fluor, and 2 1mm colonies.
E1 P104 + BIG GPC		Kan	Lots of small 0.25mm colonies	100 0.5-1mm colonies, no fluor.
E1 P104 + BIG PPC		Kan	0 colonies	1 0.5mm colony
E1 P104 + BIG PPC 6:2		Kan	0 colonies	0
E1 P104 Dephos only		Kan	1 colony on edge of plate 1.5 mm	2mm colony on edge, and 3 0.5-1mm colonies.
puc 19 (+) ctrl		Amp	100-200 1.5mm colonies	N/a
Kan (-) ctrl		Kan	0	1 1mm colony
Amp (-) ctrl		Amp	0	N/a

Restreaked some non-fluor colonies from RBS ligations/ transformations to see if repressor is being repressed-- grew in 37 and 42 degrees. No perceived difference in fluor/ no induction.

Colony PCR of Ligations 8/8

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	E1 P63 (Buffer 2) + RBS GP
	3	E1 P63 (Buffer 2) + BIG PPC A
	4	E1 P63 (Buffer 2) + BIG PPC B
	5	E1 P63 (Buffer 2) + BIG PPC C
	6	E1 P63c +BIG PPC A
	7	E1 P63c +BIG PPC B
	8	E1 P63c +BIG PPC C
	9	E1 P104 + RBS GP 6:2 A
	10	E1 P104 + RBS GP 6:2 B
	11	E1 P104 + RBS GP 6:2 C
	12	E1 P104 + RBS GP A

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	E1 P104 + RBS GP B
	2	E1 P104 + RBS GP C
	3	E1 P104 + RBS GP D
	4	E1 P104 + RBS PP A
	5	E1 P104 + RBS PP B
	6	E1 P104 + RBS PP C
	7	1 kB ladder
	8	E1 P104 + RBS PP D
	9	E1 P104 + RBS PP E
	10	E1 P104 + RBS PP F
	11	E1 P104 + RBS GPC A
	12	E1 P104 + RBS GPC B

3 bands in second gel indicate re-ligated vector.

Ligation of P104 and P63+P18 with cI RBS from TOPO

Digestion of cI RBS from TOPO and P18+P63 8/8

P18+ P63 not confirmed by sequencing yet.

'	cI857 RBS TOPO ES 1	cI857 RBS TOPO ES 2	cI857 RBS TOPO ES 3	cI857 RBS TOPO ES 5	P18+ P63 ES 1	P18+ P63 ES 2	P18+ P63 ES 3	P18+ P63 ES 4	E1 P63 + RBS PP B	E1 P104 + RBS PP A	E1 P63c + RBS PP G
DNA	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in
100X BSA	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL	0.25 uL
10X Buffer 2	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL	2.5 uL of Buffer 3	2.5 uL of Buffer 3	2.5 uL of Buffer 3
EcoRI	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL	1 uL XbaI	1 uL XbaI	1 uL XbaI
Enzyme 2	1 uL SpeI	1 uL SpeI	1 uL SpeI	1 uL SpeI	1 uL XbaI	1 uL XbaI	1 uL XbaI	1 uL XbaI	1 uL PstI	1 uL PstI	1 uL PstI
Water	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in	Fill in
Volume	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL	25 uL

Gel results 8/9

	1% agarose gel visualized using EtBr/UV
	Lane	Sample
	1	1 kB Ladder
	2	cI857 RBS TOPO ES 1
	3	cI857 RBS TOPO ES 2
	4	cI857 RBS TOPO ES 3
	5	cI857 RBS TOPO ES 5
	6
	7	1 kB Ladder
	8	P18+ P63 EX 1
	9	P18+ P63 EX 2
	10	P18+ P63 EX 3
	11	P18+ P63 EX 4
	12	1 kB Ladder
	13	E1 P63 + RBS PP B
	14	E1 P104 + RBS PP A
	15	E1 P63c + RBS PP G

RBS TOPO 1,3, and 5, and P18+ P63 EX 1-4 were extracted and purified.

Update: At least P18+ P63 1-2 have been sequence confirmed.

Digest of P104 and P63 8/9

'	P63 EX	P104 EX
DNA	5 uL	15 uL
100X BSA	0.25 uL	0.25 uL
10X Buffer	2.5 uL of Buffer 2	2.5 uL of Buffer 2
EcoRI	1 uL	1 uL
Enzyme 2	1 uL XbaI	1 uL XbaI
Water	15.25 uL	5.25 uL
Volume	25 uL	25 uL

Gel of Digestion 8/10

Extracted and purified 8/10.

mtrB, P97, P63 digests 08/06

We digested mtrB with ES and XP so that we can put it into the P63 (terminator) and P97 (RBS) vectors. We also digested P97 with SP (and dephosphorylated it).

Ligation transformation results 08/07

Plasmid	Ligation time	Ligation ratio (vector:insert)	Strain	No. of Colonies
mtrB not BB+P1 not BB old	10 min	2:6	DH5-alpha	1
mtrB not BB+P1 not BB	10 min	2:6	DH5-alpha	4
mtrB not BB+P1 not BB	5 min	2:6	TOP10	88
mtrB not BB+P3 not BB old	10 min	2:6	DH5-alpha	0
mtrB not BB+P3 not BB	10 min	2:6	DH5-alpha	8
mtrB not BB+P3 not BB	5 min	2:6	TOP10	40
mtrB+P63	10 min	2:6	TOP10	48
mtrB+P63	10 min	1:7	DH5-alpha	5
mtrB+P97	10 min	2:6	DH5-alpha	TMTC
mtrB+P97	10 min	1:7	DH5-alpha	200
P105+P1 not BB old	10 min	2:6	DH5-alpha	5
P105+P1 not BB	10 min	2:6	DH5-alpha	1
P105+P1 not BB	5 min	2:6	TOP10	2
P105+P3 not BB old	10 min	2:6	DH5-alpha	1
P105+P3 not BB	10 min	2:6	DH5-alpha	8
P105+P3 not BB	5 min	2:6	TOP10	55

Colony PCR

Ladders (end of each block) alternate as low range (100, 200, 400, 800, 2000bp) and 1kb plus. Expected band sizes indicated. (Lanes rearranged from 2% agarose 96 well E-gel)

1-16

Lane 1: Plasmid control P85 (2.3kb)
Lane 2: Colony control P88 (2.2kb)
Lanes 3-16: P105+P3 ligation colony PCR (2.5kb)

4, 6, 14 look like they have potentially correct products, so liquid cultures were grown up.

14 is incorrect- it's just P1 in pSB3K3.

17-24: P105+P1 ligation colony PCR (2.5kb)

None appear to be correct.

25-48: mtrB+P63 ligation colony PCR (2.2kb)

27, 28 picked.

RE digests 08/09

Lane 1: 1 kb ladder

Lane 2: mtrB+63 #28 cut XP (~2200+3.1kb)

Lane 3: E1P3A-P38+51C cut SP (4155)

Lane 4: E1P3A-P38+51D cut SP (4155)

Lane 5: E1P3A-P38+51B cut SP (4155)

49-64: mtrB+P97 ligation colony PCR (2.1kb)

51 picked.

65-80: mtrB'+P3 ligation colony PCR (2.3kb)

65, 69, 70, 78, 79, 80 picked.

65 sequence did not read properly.

81-96: mtrB'+P1 ligation colony PCR (2.3kb)

82, 87, 95 picked.

82 is wrong- mystery sequence. 87 and 95 are BLAST to pHELLSGATE ?!

RE digests 08/07

Lane 1: 1 kb ladder

Lane 2: E1P1+P17A cut EX (3652)

Lane 3: E1P1+P17C cut EX (3652)

Lane 4: E1P1+P17D cut EX (3652)

Lane 5: E2P1+P17A cut EX (3652)

Lane 6: E2P1+P17B cut EX (3652)

Lane 7: P108 cut SP (4155)

Lane 8: P45 cut XP (876; 2079)

Ligations 08/08

We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 2:6. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.

Transformation results 08/09

Plasmid	Amount of DNA	Strain	Ligation ratio (vector:insert by volume)	Number of colonies
P108 (vector control)	7 ul	100 ul DH5-alpha	2 (vector)!6 (water)	0
P45+108	7 ul	100 ul DH5-alpha	2:6	1
P17 in p15A (vector control)	7 ul	100 ul DH5-alpha	1 (vector):7 (water)	188
(P17 in p15A)+P39	7 ul	50 ul TOP10	1:7	1
(P17 in p15A)+P39	7 ul	100 ul DH5-alpha	1:7	0
(P17 in p15A)+P38	7 ul	50 ul TOP10	1:7	3
(P17 in p15A)+P38	7 ul	100 ul DH5-alpha	1:7	0

Colony PCR 8/9

We picked all of the noncontrol colonies and one control colony for colony PCR using the BBp/sfx primers.

	1.2% E-gel visualized using EtBr/UV
	Lane	Contents (approx. expected band size)
	1	P39+P17 in P1/3- col#1 (~940bp)
	2-5	P38+P17 in P1/3- col#2-5 (~940bp)
	6	1kb ladder
	8-9	P108+P45 (same sample) (~2.3kb)
	10	ligation control (no insert)

Since the bands are at least plausible, the P38/39 in P1/3 samples were grown up and miniprepped.

Retransformation 8/9

We retransformed 5μL of the P39+17 in P1/3 ligation into 50μL DH5α and obtained two colonies.

Colony PCR shows that one of these does not contain the right construct. The other was set up in 5ml culture.

1kb ladder, colony A, colony B (both should have ~950 products if correct)

New ligations and transformations 08/10

We retransformed our old P108+45 (2:6 vector to insert) ligation and the old vector control. We also did this ligation over with a 2:1 molecular ratio of insert to vector. Both P108 (4155 bp) and P45 (876 bp) were about 17 ng/μL. We used a ratio of 5.4:2.3 vector to insert by volume.

Transformation results

Plasmid	Ratio (vector!insert)	Strain	Number of colonies
P108 vector control (old)	2 ul P108!6 ul EB	DH5-alpha	0
P108+45 (old)	2!6	DH5-alpha	0
P108+45 (old)	2!6	TOP10	4
P108 vector control (new)	5.4 ul P108!2.3 ul EB	DH5-alpha	0
P108+45 (new)	5.4 ul P108!2.3 ul EB	DH5-alpha	2
P108+45 (new)	5.4 ul P108!2.3 ul EB	DH5-alpha	8

Transforming LacZ parts

We attempted to transform the following parts: I732017, E0435, E0435, I732005 into our competent DH5α. E0435 (on CM) had 0 colonies). The others (on Carb) had 10-20 extremely small colonies (after 16+hrs of incubation) each. We picked 2 of each for liquid culture, but none grew.

We requested the parts directly from MIT.

Team:Harvard/Dailybook/Week7/Chemical and Light

From 2008.igem.org

Contents

PCR

8/4: Cph/EnvZ, P1, P3, mtrB not BB

8/4 Colony PCR of putative mtrB samples

8/4 CphEnvZ, P1, P3, mtrB

8/5: gels

Ligations and transformations 08/06

TOPO cloning

08/04

P1, mtrB

ompR

HO-pcyA

Transforming in XL10-Gold cells

Ligations

HO-pcyA, ompR, CDF

Colony PCR 8-6

Digests, 1-8

9-19

20-30

31-41

42-52

53-63

64-74

75-85

86-94, P20 controls

Making Thermoinducible cI System

Ligation from 8/1

Analytical Digest Gels from TOPO Cloning 8/5

Digestion and Ligation of P18+P63 and P75+P63

Digestion of P18, P63, and P75 8/4

Gel of Digestion 8/5

Ligation of P18+P63 and P75+P63 8/5

Plate Results 8/6

Digestion and Ligation of P63 + cI857 (Both Primer Sets)

Digestion of cI857 PCR product and P63 8/5

Gel results 8/6

Ligation of P63, P63c, and P104 with cI857 8/6

Plate results 8/7

Colony PCRs of 8/6 Ligations 8/7

Ligation of P63, P63c, and P104 with cI (repeat) 8/7

Digestions of P63, P63c, and P104 8/6

Gel of Digestion 8/7

Ligation of P63, P63c, and P104 with cI 857 8/7

Plate Results 8/8

Colony PCR of Ligations 8/8

Ligation of P104 and P63+P18 with cI RBS from TOPO

Digestion of cI RBS from TOPO and P18+P63 8/8

Gel results 8/9

Digest of P104 and P63 8/9

Gel of Digestion 8/10

mtrB, P97, P63 digests 08/06

Ligation transformation results 08/07

Colony PCR

1-16

17-24: P105+P1 ligation colony PCR (2.5kb)

25-48: mtrB+P63 ligation colony PCR (2.2kb)

RE digests 08/09

49-64: mtrB+P97 ligation colony PCR (2.1kb)

65-80: mtrB'+P3 ligation colony PCR (2.3kb)

81-96: mtrB'+P1 ligation colony PCR (2.3kb)

RE digests 08/07

Ligations 08/08

Transformation results 08/09

Colony PCR 8/9

Retransformation 8/9

New ligations and transformations 08/10

Transformation results

Transforming LacZ parts