"

From 2010.igem.org

Labwork

Last week we did 2 DLTs, but both resulted in empty plates (no colonies). Apparently something went wrong, we think it was the diluted ligase that lost its activity. So, today we will try to do the DLT again, but then with a different ligase.

Digestion

#	Sample	Enzyme 1	Enzyme 2	Extra	Buffer	BSA
1	018A	EcoRI	SpeI	AseI	2 (Biolabs)	✓
2	019A	EcoRI	XbaI	Phosphatase	2 (Biolabs)	✓
3	J13002	SpeI	PstI	Phosphatase	2 (Biolabs)	✓
4	ladA	XbaI	PstI	AseI	2 (Biolabs)	✓
5	009A	EcoRI	SpeI	AseI	2 (Biolabs)	✓
6	010A	EcoRI	XbaI	Phosphatase	2 (Biolabs)	✓
7	104T	XbaI	PstI	✗	2 (Biolabs)	✓
8	PalkB	SpeI	PstI	✗	2 (Biolabs)	✓
9	327	SpeI	PstI	✗	2 (Biolabs)	✓
10	K1	XbaI	PstI	AseI	2 (Biolabs)	✓
11	GFP-TT	XbaI	PstI	AseI	2 (Biolabs)	✓

Ligation

#	BioBrick	Fragment 1	Recipient Vector/fragment 2
1	026	E - J61101 - ADH - S (018)	E - pSB1A2 - J61107 - ALDH - X (019)
2	027	X - ladA - P	P - pSB1A2 - J13002 - S
3	028	E - J61100 - rubA4 - S (009)	X - J61100 - rubR - pSB1A2 - E (010)
4	108	X - BBC1 - B0015 - P	P - pSB1A2 - J13002 - S
5	306	X - K1 - P	P - pSB1A2 - PalkB - S
6	331	P - pSB1A2 - 327 - S	X - GFP-TT - P

The ligation mixtures were ligated for 1 hour, after which they were added to competent K12 E.coli cells and plated on LB-agar with ampicillin.