IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week4/Chemical and Light

Primers

HO+pcyA, P3 vector w/o GFP

Primers were designed to PCR out the heme oxygenase and pcyA coding regions from the UT Austin plasmids. Due to the RE sites in these nonBB plasmids, their ends are given ApaLI and KpnI sites. The same is done for the P3 vector (the primers are such that we can PCR out the backbone, along with the RBS and terminator. The "P3-GFP" primers should also work for P1. However, I'm a little concerned, b/c the annotation of the P1 and P3 vectors disagrees with the sequence given for the RBS and terminator subparts.

P38, P39 Oligos

5' phospho oligos were made to anneal into what is effectively P38 and P39 digested with ES.

Ligations

Results from 7/12 ligations (transformed into E1)

DNA	Selection used on plate	# colonies	Notes
17XP+38SP	AMP	2 AFTER 48HRS	Plate put in 4°C, not picked
63+59 FROM 7/9 LIGATION	KAN	1 AFTER 48HRS	Plate put in 4°C, not picked
CDF XP (7/4)+P1XP	KAN	1 AFTER 48HRS	Plate put in 4°C, not picked
45XP(7/11 45)+39SP	AMP	0
45XP(7/11 45)+38SP	AMP	0
77XP+38SP	AMP	0
52XP+39SP	AMP	0
77XP+39SP	AMP	0
89XP+38SP	AMP	0
45XP+39SP	AMP	0
89XP+39SP	AMP	0
51XP+39SP	AMP	0
17XP+39SP	AMP	0
P88	KAN	0
63+58 FROM 7/9 LIGATION	KAN	0
58EX+63ES	KAN	0
52XP+38SP	AMP	0
89ES+45EX	CARB	TMTC	The 45 EX used was not purified, so the miniprep culture was of religated P45 and did not glow. However, we picked fluorescent colonies from the plate and restreaked/set up liquid cultures of them.
CDF XP(7/4)+P1XP(7/1)	KAN	68	miniprepped
17ES+45EX	CARB	TMTC	The 45 EX used was not purified, so the miniprep cultures were of religated P45 and did not glow. However, we picked fluorescent colonies from the plate and restreaked/set up liquid cultures of them.
45XP+38SP	AMP	4	DID NOT FLUORESCE- not miniprepped
51XP+38SP	CARB	5	2 picked colonies, neither grew- repicked 7/14, these did not grow either
54ES+59EX	KAN	3	DID NOT FLUORESCE- not miniprepped
CDF XP(7/12)+P1XP(7/1)	KAN	TMTC	miniprepped
CDF XP(7/12)+P1	KAN	232	miniprepped
no DNA	AMP	0
no DNA	KAN	0
no DNA	CARB	6 big + 10 small
no cells	KAN	0
1μL pUC19	CARB	96	did grow in LB AMP medium (+ control)
1μL pUC19	AMP	64

We retransformed all of these on 07/15 using 5 μL DNA for 50 μL DH5α cells. We put the cells directly into liquid cultures after transforming them.

RE digests 07/15

We digested P53/54 with ES and P58/59 with EX. We intend to ligate these parts together. This will give us RBS + TetR/LacI + terminator + pTet/pLac + RBS + GFP + terminator.

We also digested P90 with SP and P48/P49 with XP. This will give us the CDF origin in a p15A vector with an Amp or Cm resistance cassette.

We followed the standard digest protocol under the "General Protocols" section.

Gel 1

Lane 1: 1 kb ladder

Lane 2: P48 cut XP (769 bp)

Lane 3: P48 uncut (3477 bp)

Lane 4: P49A cut XP (943 bp)

Lane 5: P49A uncut (3651 bp)

Lane 6: P49B cut XP (943 bp)

Lane 7: P49B uncut (3651 bp)

Lane 8: P49C cut XP (943 bp)

Lane 9: P49C uncut (3651 bp)

Lane 10: P53 cut ES (840 bp)

Gel 2

Lane 1: P53 uncut (2919 bp)

Lane 2: P54 cut ES (1308 bp)

Lane 3: P54 uncut (3387 bp)

Lane 4: P58 cut EX (~3016 bp)

Lane 5: P58 uncut (3016 bp)

Lane 6: P59A cut EX (~3201 bp)

Lane 7: P59A uncut (3201 bp)

Lane 8: P59B cut EX (~3201 bp)

Lane 9: 1 kb ladder

Gel 3

Lane 1: P59B uncut (3201 bp)

Lane 2: P59C cut EX (~3201 bp)

Lane 3: P59C uncut (3201 bp)

Lane 4: P90A cut SP (~3650 bp)

Lane 5: P90A uncut (~3650 bp)

Lane 6: P90A cut SP (~3650 bp)

Lane 7: P90A uncut (~3650 bp)

Lane 8: P90A cut SP (~3650 bp)

Lane 9: P90A uncut (~3650 bp)

Lane 10: blank

Lane 11: 1 kb ladder