Berk2010-Group

Amy N. Kristofferson 17:15, 7 June 2010 (EDT)

First step of Manual Assembly

Ligation:<br> Followed this protocol with the following amendments:

Mastermix 
*6.5uL ddH2O
*1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
*0.5uL T4 DNA Ligase
DNA
*3uL Lefty vector
*3uL Right vector

Zymo: Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.

Digestion: Lefty MM added to 9+8, 7+11, 9+20
Right MM added to 7+23, 8+6, 7+5
They should be done incubating at 3:48pm.

Lefty MasterMix (for 4uL miniprep DNA)

• 4uL water
• 1uL of NEB2
• 0.5uL XhoI
• 0.5uL BamH1

Righty MasterMix (for 4uL miniprep DNA)

• 4uL water
• 1uL of NEB2
• 0.5uL XhoI
• 0.5uL BglII

Christoph Neyer 16:54, 7 June 2010 (EDT)

Some of Tim's spreadsheet data (which clones have been chosen for 2ab assembly):

								Chosen clone (June2)		Chosen clone (June3)
	4	AC	9	B10sbb20	<FokI-!		1	1		
	4	AC	10	B10sbb43	{rbs2_lamB>}		2	1		
	4	AC	16	B10sbb15	phiC31 attB		2	1	seq	
	4	AC	17	B10sbb14	rbs_phiC31>		3	1		
5	4	AC	18	B10sbb13	<phiC31>		3	1		
	7	AK	2	B10sbb09	Tn5 3'TR		4	1	seq	
	7	AK	5	B10sbb06	piggieBac3'TR		4	1		
	7	AK	9	B10sbb20	<FokI-!		5	1		
	7	AK	10	B10sbb43	{rbs2_lamB>}		5			
	7	AK	11	Bjh1906	b1006		6	1	seq	
	7	AK	13	B10sbb18	<zf->		7	1		
	7	AK	14	B10sbb17	<zf+>		8	1		
	7	AK	19	B10sbb12	sleeping_beauty_3'TR		8	1		
	7	AK	23	B10sbb42	{rbs_pelB>}		9			d	
10	7	AK	24	Bjh2294	Self_Lysis_Device		9			c	
	9	CA	3	Bjh1858	<NLS!		10	1	seq		
	9	CA	8	ig114	{AraC-Pbad}		11	1			
	9	CA	11	Bjh1906	b1006		11			a	seq
	9	CA	13	B10sbb18	<zf->		11	1			
	9	CA	20	B10sbb44	Promoter_rbs_GenR		12	2			
6	9	CA	23	B10sbb42	{rbs_pelB>}		13			a	seq
	5	CK	3	Bjh1858	<NLS!		13	1	seq		
	5	CK	8	ig114	{AraC-Pbad}		13	1			
	5	CK	15	B10sbb16	phiC31 attP		14	1	seq		
	5	CK	20	B10sbb44	Promoter_rbs_GenR		15	2			
5	5	CK	23	B10sbb42	{rbs_pelB>}		16	1	seq		
	6	KA	2	B10sbb09	Tn5 3'TR		17	2	seq		
	6	KA	4	B10sbb07	<tn5>		17	2			
	6	KA	5	B10sbb06	piggieBac3'TR		18	2			
	6	KA	7	B10sbb04	<piggyBac!		18	2		
	6	KA	11	Bjh1906	b1006		19	1	seq	
	6	KA	12	B10sbb19	<FokI+!		19	2	seq	
	6	KA	13	B10sbb18	<zf->		20	2		
	6	KA	19	B10sbb12	sleeping_beauty_3'TR		20	2		
	6	KA	22	B10sbb10	<SB100x!		21	1		
	6	KA	23	B10sbb42	{rbs_pelB>}		22	2		
11	6	KA	24	Bjh2294	Self_Lysis_Device		23			a
	8	KC	1	B10sbb08	Tn5 5'TR		23	1	seq	
	8	KC	4	B10sbb07	<tn5>		23	2		
	8	KC	6	B10sbb05	piggieBac_5'TR		23	1		
	8	KC	17	B10sbb14	rbs_phiC31>		24	2		
	8	KC	18	B10sbb13	<phiC31>		24	1		
6	8	KC	21	B10sbb11	sleeping_beauty_5'TR		24	1