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)

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, P3, mtrB

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

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