Berk2010-Group

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

First step of Manual Assembly

Ligation:
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		

Tim Hsiau 19:48, 2 June 2010 (EDT)

Tim Hsiau 16:16, 1 June 2010 (EDT)

EcoXho's for the first round of assembly (ecobammed into assembly vectors)

appropriate fragment lengths

layout on ecoXho gel		FRAGMENTS		part size
1	6-2	1307	1605	35
2	6-11	1318	1605	46
3	6-2	1307	1605	35
4	6-11	1318	1605	46
5	6-4	2703	1605	1431
6	6-12	1869	1605	597
7	6-4	2703	1605	1431
8	6-12	1869	1605	597
9	6-5	1513	1605	241
10	6-13	1671	1605	399
11	6-5	1513	1605	241
12	6-13	1671	1605	399
13	6-7	3060	1605	1788
14	6-19	1500	1605	228
15	6-7	3060	1605	1788
16	6-19	1500	1605	228
17	6-22	2298	1605	1026
18	7-9	1797	1680	597
19	6-22	2298	1605	1026
20	7-10	1300	1680	100
21	6-23	1363	1605	91
22	7-11	1246	1680	46
23	6-23	1363	1605	91
24	7-13	1599	1680	399
25	6-24	#N/A	1605	#N/A
26	7-14	1599	1680	399
27	6-24	#N/A	1605	#N/A
28	7-19	1428	1680	228
29	7-2	1235	1680	35
30	7-23	1291	1680	91
31	7-5	1441	1680	241
32	7-24	#N/A	1680	#N/A
33	7-24	#N/A	1680	#N/A
34	5-23	1291	1473	91
35	5-3	1233	1473	33
36	5-23	1291	1473	91
37	5-3	1233	1473	33
38	8-1	1307	1473	35
39	5-8	2444	1473	1244
40	8-1	1307	1473	35
41	5-8	2444	1473	1244
42	8-4	2703	1473	1431
43	5-15	1290	1473	90
44	8-4	2703	1473	1431
45	5-20	2023	1473	823
46	8-6	1591	1473	319
47	5-20	2023	1473	823
48	8-6	1591	1473	319
				
				
				
1	8-17	3117	1473	1845
2	4-10	1166	1679	100
3	8-17	3117	1473	1845
4	4-10	1166	1679	100
5	8-18	3090	1473	1818
6	4-16	1156	1679	90
7	8-18	3090	1473	1818
8	4-16	1156	1679	90
9	8-21	1504	1473	232
10	4-17	2911	1679	1845
11	8-21	1504	1473	232
12	4-17	2911	1679	1845
13	4-9	1663	1679	597
14	4-18	2884	1679	1818
15	4-9	1663	1679	597
16	4-18	2884	1679	1818
17	9-3	1099	1605	33
18	9-20	1889	1605	823
19	9-3	1099	1605	33
20	9-20	1889	1605	823
21	9-8	2310	1605	1244
22	9-23	1157	1605	91
23	9-8	2310	1605	1244
24	9-23	1157	1605	91
25	9-11	1112	1605	46
26				
27	9-11	1112	1605	46
28				
29	9-13	1465	1605	399
30				
31	9-13	1465	1605	399
32				
33				
34				
35