Team:TU Delft/16 August 2010 content

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Revision as of 18:36, 16 August 2010

Alkane degradation

Unfortunately last week's attempts hadn't yielded any BioBricks making use of the 2-way ligation method. We thus decided to take another stab at the 3-way ligation procedure, according to the BioBrick Assembly Manual of Ginkgo Bioworks. The BioBrick numbers we tried to construct were: 014C, 020C, 405C, 327C and 304C.

Digestion

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	011A	EcoRI	SpeI	AseI	NEBuffer 2	✓	‘E - J23100-J61100-alkB2-J61100-rubA3 - S’
2	013K	XbaI	PstI	HindIII	NEBuffer 2	✓	‘X - J61100-rubA4-J61100-rubR-B0015 - P’
3	017A	EcoRI	SpeI	AseI	NEBuffer 2	✓	‘E - J23100-J61100-ladA - S’
4	018A	XbaI	PstI	AseI	NEBuffer 2	✓	‘X - J61101-ADH - P’
5	402A	EcoRI	SpeI	AseI	NEBuffer 2	✓	‘E - J23100-J61101-PhPFDα - S’
6	403A	XbaI	PstI	AseI	NEBuffer 2	✓	‘X - J61101-PhPFDβ - P’
7	pCaiF	EcoRI	SpeI	AseI	NEBuffer 2	✓	‘E - pCaiF - S’
8	B0032	XbaI	PstI	AseI	NEBuffer 2	✓	‘X - B0032 - P’
9	B0032	XbaI	PstI	AseI	NEBuffer 2	✓	‘X - B0032 - P’
10	pAlkS	EcoRI	SpeI	AseI	NEBuffer 2	✓	‘E - pAlkS - P’
11	pSB1C3	EcoRI	PstI	None	NEBuffer 2	✓	‘E - pSB1C3 - P’

The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:

1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker

@@ Line 116: / Line 116: @@
 The digestions were executed for 15 minutes at 37 degrees, and checked on [https://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
+[[Image:TU_Delft_Digestion_16-08-10.png|400px|thumb|left|1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
-[[Image:|300px|thumb|left|TU_Delft_Digestion_16-08-10]]