IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light

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

8/5: gels

Gel for excision (includes old PCR samples)

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

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

75-85

	1% agarose 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% agarose 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

[Gels here]

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

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

Used dephosphorylated and undephosphorylated vector.

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

Done with gel purified and pcr purified cI857, and in tandem with Christina.

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

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)