Berk2010-Group

First step of Manual Assembly
Ligation: Followed this protocol with the following amendments: Mastermix DNA
 * 6.5uL ddH2O
 * 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 * 0.5uL T4 DNA Ligase
 * 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	}		2	1 4	AC	16	B10sbb15	phiC31 attB		2	1	seq 4	AC	17	B10sbb14	rbs_phiC31>		3	1 5	4	AC	18	B10sbb13			3	1 7	AK	2	B10sbb09	Tn5 3'TR		4	1	seq 7	AK	5	B10sbb06	piggieBac3'TR		4	1 7	AK	9	B10sbb20	}		5 7	AK	11	Bjh1906	b1006		6	1	seq 7	AK	13	B10sbb18			7	1 7	AK	14	B10sbb17			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			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	}		16	1	seq 6	KA	2	B10sbb09	Tn5 3'TR		17	2	seq 6	KA	4	B10sbb07	 		17	2 6	KA	5	B10sbb06	piggieBac3'TR		18	2 6	KA	7	B10sbb04			20	2 6	KA	19	B10sbb12	sleeping_beauty_3'TR		20	2 6	KA	22	B10sbb10	}		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	 		23	2 8	KC	6	B10sbb05	piggieBac_5'TR		23	1 8	KC	17	B10sbb14	rbs_phiC31>		24	2 8	KC	18	B10sbb13			24	1 6	8	KC	21	B10sbb11	sleeping_beauty_5'TR		24	1

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