Berk2010-Christoph

Christoph Neyer 16:08, 14 July 2010 (EDT)

Checking some of the parts from Oliver twist:

Layout:
| align="center" style="background:#f0f0f0;"|Lane # | align="center" style="background:#f0f0f0;"|' | align="center" style="background:#f0f0f0;"|Plate | align="center" style="background:#f0f0f0;"|Location | align="center" style="background:#f0f0f0;"|part | align="center" style="background:#f0f0f0;"|Part size | align="center" style="background:#f0f0f0;"|Eco/xho Frags | align="center" style="background:#f0f0f0;"|eco/Xho Frags |- | 1||sbb15 in AC||Future||A1||sbb15||90||1156||1679 |- | 3||sbb43 in AK||Future||A4||sbb43||100||1300||1680 |- | 5||Bjh1906 in AK||Future||B4||Bjh1906||47||1247||1680 |- | 7||sbb13 in AC||Future||B7||sbb13||1818||2884||1679 |- | 9||Bjh2294 in AK||Future||F10||Bjh2294||2507||3707||1680 |- | 11||Bjh1858 in CA||Future||A12||Bjh1858||33||1305||1473 |- | 2||sbb15 in AC||Tiny Tim||A1||sbb15||90||1156||1679 |- | 4||sbb43 in AK||Tube C||na||sbb43||100||1300||1680 |- | 6||Bjh1906 in AK||Cat||A1||Bjh1906||47||1247||1680 |- | 8||sbb13 in AC||Tiny Tim||C7||sbb13||1818||2884||1679 |- | 10||Bjh2294 in AK||Cat||A5||Bjh2294||2507||3707||1680 |- | 12||Bjh1858 in CA||Scrooge||A12||Bjh1858||33||1305||1473 |}

Christoph Neyer 18:05, 13 July 2010 (EDT)

Rechecked pMLL7 and pMLL4 for methylated parts:

Layout:

Lane #	Part in vector	opposite vector	Part	Vector	Part	Location in Tiny Tim	Enzymes	Expected bands	Expected bands	Wrong Bands	Wrong Bands
1	Bjh2294 in AK	pMLL6-KA	Bjh2294	pMLL7-AK	Bjh2294	Get from Chesire cat A 5	EcoRI/Bgl1	4554	830	3479	1905
2	Bjh1906 in CA	pMLL4-AC	Bjh1906	pMLL9-CA	Bjh1906	A6	EcoRI/Bgl1	1773	1019	1962	830
3	sbb06 in AK	pMLL6-KA	sbb06	pMLL7-AK	sbb06	G10	EcoRI/Bgl1	2288	830	1905	1213
4	sbb44 in CA	pMLL4-AC	sbb44	pMLL9-CA	sbb44	C6	EcoRI/Bgl1	1795	1773	2738	830

Christoph Neyer 23:23, 12 July 2010 (EDT)

Stage 1 Round 3 RE map of minipreps for parts not involving KA inputs. Located on Oliver Twist. Gel Part A:

Gel Part B:

Layout:

RE Map of Oliver Twist	Part A Lane #	Part B Lane #	Part C Lane #	Part D Lane #	Part Name	Part size	Lefty size	Righty Size	Eco/xho Frags	eco/Xho Frags	Analysis A	Analysis B	Re-run/re-pick?
iGEM10_076 in AK	1	2	23	24	iGEM10_076	1878	1845	33	3078	1680
iGEM10_055 in KA	3	4	25	26	iGEM10_055	1464	1431	33	2736	1605
iGEM10_068 in AK	5	6	27	28	iGEM10_068	771	681	90	1971	1680
iGEM10_065 in CK	7	8	29	30	iGEM10_065	2554	47	2507	3754	1473
iGEM10_073 in CK	9	10	31	32	iGEM10_073	996	399	597	2196	1473
iGEM10_054 in CK	11	12	33	34	iGEM10_054	1335	1244	91	2535	1473
iGEM10_057 in CK	13	14	35	36	iGEM10_057	858	823	35	2058	1473
iGEM10_061 in CK	15	16	37	38	iGEM10_061	1064	823	241	2264	1473
iGEM10_063 in CK	17	18	39	40	iGEM10_063	1057	823	234	2257	1473
iGEM10_074 in CK	19	20	41	42	iGEM10_074	490	91	399	1690	1473
iGEM10_055 in KA	21	22	43	44	iGEM10_055	1464	1431	33	2736	1605

Christoph Neyer 19:24, 12 July 2010 (EDT)

Col PCR Stage 1 Round 3:
Part A:

Part B:

Part C:

Christoph Neyer 16:41, 12 July 2010 (EDT)

Checking Vector Backbones Gel:

Lane #	Part in vector	Vector + Ebid	Location in Romeo	Enzymes	Expected bands	Expected bands	Wrong Bands	Wrong Bands
1	sbb14 in AC	4 + 17	E8	EcoRI/Bgl1	3760	830	2817	1773
2	ig114 in CK	5 + 8	D5	EcoRI/ScaI	3221	694	2146	1769
3	sbb06 in KA	6 + 5	F1	EcoRI/Bgl1	1905	1213	2288	830
4	sbb17 in AK	7 + 14	E4	EcoRI/Bgl1	2446	830	1905	1371
5	sbb07 in KC	8 + 4	F6	EcoRI/ScaI	2333	1769	3408	694
6	sbb18 in CA	9 + 13	G9	EcoRI/Bgl1	1773	1371	2314	830

Christoph Neyer 21:25, 7 July 2010 (EDT)

Did colony PCR of Stage 1 Round 2assembly parts.

• Gel #1
  • Part A:
    
  • Part B:
    

• Gel #2
  • Part A:
    
  • Part B:
    

Layout for both gels:

'	Lane	'	'
	Clone 1,3	Clone 2,4	Expected Band
iGEM10_076 in AK	1	2	2078
iGEM10_055 in KA	3	4	1664
iGEM10_068 in AK	5	6	971
iGEM10_064 in KC	7	8	1196
iGEM10_065 in CK	9	10	2754
iGEM10_056 in AC	11	12	282
iGEM10_057 in CA	13	14	1058
iGEM10_060 in AC	15	16	566
iGEM10_062 in AC	17	18	479
iGEM10_066 in CA	19	20	280
iGEM10_061 in CA	21	22	1264
iGEM10_063 in CA	23	24	1257
iGEM10_054 in CK	25	26	1535
iGEM10_057 in CK	27	28	1058
iGEM10_061 in CK	29	30	1264
iGEM10_063 in CK	31	32	1257
iGEM10_071 in KC	33	34	2109
iGEM10_072 in KC	35	36	897
iGEM10_073 in CK	37	38	1196
iGEM10_074 in CK	39	40	690
iGEM10_075 in CK	41	42	690
iGEM10_055 in KA	43	44	1664
iGEM10_054 in CA	45	46	1535
iGEM10_067 in CA	47	48	357

Christoph Neyer 19:35, 7 July 2010 (EDT)

Col PCR of oparts 77-79:

Christoph Neyer 21:47, 6 July 2010 (EDT)

Did round 1 of 2ab assembly today. Here's the gel for ColPCR of 7 with 10 into Lefty strain.

Christoph Neyer 16:31, 5 July 2010 (EDT)

Restriction map of Methylated Parts for stage 0 reruns:

Layout:

Re-runs	'	'	'	'	'	'
sbb43 in AK	1	2	sbb43	100	1300	1680
ig114 in CA	3	4	ig114	1244	2516	1473
Bjh1858 in CK	5	6	Bjh1858	33	1233	1473
sbb07 in KA	7	8	sbb07	1431	2703	1605
sbb20 in AK	9	10	sbb20	597	1797	1680

Christoph Neyer 20:41, 2 July 2010 (EDT)

Restriction maps of Methylated Parts for Stage 0:

• Lefty methylations:
  
  • 
  • 
  • Layout:
    

'	Part A Lane #	Part B Lane #	Part Name	Part size	Eco/xho Frags	eco/Xho Frags
sbb15 in AC	1	2	sbb15	90	1156	1679
sbb14 in AC	3	4	sbb14	1845	2911	1679
Bjh1882 in AC	5	6	Bjh1882	681	1747	1679
sbb19 in KA	7	8	sbb19	597	1869	1605
sbb18 in KA	9	10	sbb18	399	1671	1605
sbb10 in KA	11	12	sbb10	1026	2298	1605
sbb42 in KA	13	14	sbb42	91	1363	1605
sbb04 in KA	15	16	sbb04	1788	3060	1605
sbb42 in CK	17	18	sbb42	91	1291	1473
sbb37 in CK	19	20	sbb37	41	1241	1473
Bjh1858 in CK	21	22	Bjh1858	33	1233	1473
ig114 in CK	23	24	ig114	1244	2444	1473
Bca1091 in CK	25	26	Bca1091	60	1260	1473
sbb44 in CK	27	28	sbb44	823	2023	1473
sbb43 in AK	29	30	sbb43	100	1300	1680
Bjh1906 in AK	31	32	Bjh1906	47	1247	1680
sbb14 in KC	33	34	sbb14	1845	2911	1605
sbb13 in KC	35	36	sbb13	1818	2884	1605
sbb07 in KC	37	38	sbb07	1431	2497	1605
Bjh1906 in CA	39	40	Bjh1906	47	1319	1473
sbb18 in CA	41	42	sbb18	399	1671	1473
sbb44 in CA	43	44	sbb44	823	2095	1473
sbb42 in CA	45	46	sbb42	91	1363	1473
ig114 in CA	47	48	ig114	1244	2516	1473

• Righty Methylations:
  
  • 
  • 
  • Layout:
    

'	Part A Lane #	Part B Lane #	Part Name	Part size	Eco/xho Frags	eco/Xho Frags
sbb43 in AC	1	2	sbb43	100	1166	1679
sbb13 in AC	3	4	sbb13	1818	2884	1679
sbb20 in AC	5	6	sbb20	597	1663	1679
sbb06 in KA	7	8	sbb06	241	1513	1605
sbb04 in KA	9	10	sbb04	1788	3060	1605
Bjh2245 in KA	11	12	Bjh2245	97	1369	1605
sbb16 in CK	13	14	sbb16	90	1290	1473
Bjh1858 in CK	15	16	Bjh1858	33	1233	1473
Bjh1906 in KA	17	18	Bjh1906	47	1319	1605
sbb19 in KA	19	20	sbb19	597	1869	1605
sbb12 in KA	21	22	sbb12	234	1506	1605
sbb09 in KA	23	24	sbb09	35	1307	1605
sbb10 in KA	25	26	sbb10	1026	2298	1605
sbb42 in KA	27	28	sbb42	91	1363	1605
Bjh2294 in KA	29	30	Bjh2294	2507	3779	1605
sbb07 in KA	31	32	sbb07	1431	2703	1605
sbb18 in AK	33	34	sbb18	399	1599	1680
sbb17 in AK	35	36	sbb17	399	1599	1680
sbb12 in AK	37	38	sbb12	234	1434	1680
sbb09 in AK	39	40	sbb09	35	1235	1680
sbb42 in AK	41	42	sbb42	91	1291	1680
Bjh2294 in AK	43	44	Bjh2294	2507	3707	1680
sbb06 in AK	45	46	sbb06	241	1441	1680
sbb20 in AK	47	48	sbb20	597	1797	1680

• Extra Righties:
  
  • 
  • Layout:
    

'	Part A Lane #	Part B Lane #	Part Name	Part size	Eco/xho Frags	eco/Xho Frags
sbb08 in KC	1	2	sbb08	35	1101	1605
sbb11 in KC	3	4	sbb11	232	1298	1605
sbb05 in KC	5	6	sbb05	319	1385	1605
Bjh1858 in CA	7	8	Bjh1858	33	1305	1473

Christoph Neyer 20:18, 2 July 2010 (EDT)

Decided to redo all the methylations.

Christoph Neyer 18:40, 29 June 2010 (EDT)

Restriction digest of iGEM10_76 (clones 2-6). Expect bands at ~3100 and ~1700:

Christoph Neyer 19:03, 28 June 2010 (EDT)

Colony PCR of manually assembled parts iGEM10_076-79:

Christoph Neyer 17:45, 28 June 2010 (EDT)

Restriction map of catchup methylated parts:

Layout:

Part	Strain	Clone number	Part number	'	Lane Number	Part size	eco/xho frags	eco/xho frags
8 with 1	R	4	1		1	35	1307	1473
9 with 3	R	4	3		2	33	1099	1605
8 with 1	R	3	1		3	35	1307	1473
9 with 3	R	3	3		4	33	1099	1605
8 with 1	R	2	1		5	35	1307	1473
9 with 3	R	2	3		6	33	1099	1605
8 with 1	R	1	1		7	35	1307	1473
9 with 3	R	1	3		8	33	1099	1605
						--	--	--
9 with 23	L	4	23		9	91	1157	1605
5 with 20	L	4	20		10	823	2023	1473
9 with 23	L	3	23		11	91	1157	1605
5 with 20	L	3	20		12	823	2023	1473
9 with 23	L	2	23		13	91	1157	1605
5 with 20	L	2	20		14	823	2023	1473
9 with 23	L	1	23		15	91	1157	1605
5 with 20	L	1	20		16	823	2023	1473
Ladder						--
9 with 11	L	4	11		17	46	1112	1605
8 with 21	R	B1	21		18	232	1504	1473
9 with 11	L	3	11		19	46	1112	1605
8 with 21	R	B2	21		20	232	1504	1473
9 with 11	L	2	11		21	46	1112	1605
8 with 21	R	B3	21		22	232	1504	1473
9 with 11	L	1	11		23	46	1112	1605
8 with 21	R	B4	21		24	232	1504	1473
						--	--	--
9 with 20	L	4	20		25	823	1889	1605
8 with 21	R	A1	21		26	232	1504	1473
9 with 20	L	3	20		27	823	1889	1605
8 with 21	R	A2	21		28	232	1504	1473
9 with 20	L	2	20		29	823	1889	1605
8 with 21	R	A3	21		30	232	1504	1473
9 with 20	L	1	20		31	823	1889	1605
8 with 21	R	A4	21		32	232	1504	1473

Christoph Neyer 19:41, 25 June 2010 (EDT)

Made methylated basic parts and plated. Need to take out of incubator on Saturday.
Did manual 2ab test run for:

• iGEM10_076
• iGEM10_077
• iGEM10_078
• iGEM10_079

Plated on a strip today with four different volumes (20,30,40,50ul).

TO DO:

• Need to double check 7 w/ 10 Lefty from plate A. Looked like it didn't get digested.
  
• Need to colony PCR basic parts and composite parts on Monday and then minprep and restriction map for assembly on Tuesday.
  

Christoph Neyer 19:41, 25 June 2010 (EDT)

Made methylated basic parts and plated. Need to take out of incubator on Saturday.
Did manual 2ab test run for:

• iGEM10_076
• iGEM10_077
• iGEM10_078
• iGEM10_079

Plated on a strip today with four different volumes (20,30,40,50ul).

TO DO:

• Need to double check 7 w/ 10 Lefty from plate A. Looked like it didn't get digested.
  
• Need to colony PCR basic parts and composite parts on Monday and then minprep and restriction map for assembly on Tuesday.
  

Christoph Neyer 21:13, 24 June 2010 (EDT)

TO DO:

• Make methylated basic parts:
  • 9 with 11
  • 5 with 20
  • 9 with 20
  • 9 with 23
  • 8 with 1
  • 8 with 21
  • 9 with 3
• Manual 2ab test run for two junctions (4 basic parts -> 2 composite parts):
  • iGEM10_065
  • iGEM10_071
• Create methylated plate layout and transfer parts to it.
• Once methylated plate is made restriction map it and sequence a few.

Christoph Neyer 17:54, 24 June 2010 (EDT)

Restricton map of methylated parts from plate A:

Layout of parts from plate A:

Parts from plate A	'	'	'	'	'
Plasmid	Part number	Part Size	Eco/Xho Frags	Eco/Xho Frags	Lane #
5 with 20	20	823	2023	1473	1
5 with 25	25	41	1241	1473	2
9 with 11	11	46	1112	1605	3
9 with 20	20	823	1889	1605	4
9 with 23	23	91	1157	1605	5
7 with 10	10	100	1300	1680	6
5 with 15	15	90	1290	1473	7
5 with 3	3	33	1233	1473	8
8 with 1	1	35	1307	1473	9
8 with 21	21	232	1504	1473	10
4 with 10	10	100	1166	1679	11
4 with 18	18	1818	2884	1679	12
4 with 9	9	597	1663	1679	13
9 with 3	3	33	1099	1605	14
Ladder
6 with 11	11	46	1318	1605	15
7 with 13	13	399	1599	1680	16
6 with 12	12	597	1869	1605	17
7 with 14	14	399	1599	1680	18
6 with 19	19	228	1500	1605	19
7 with 19	19	228	1428	1680	20
6 with 2	2	35	1307	1605	21
7 with 2	2	35	1235	1680	22
6 with 22	22	1026	2298	1605	23
7 with 23	23	91	1291	1680	24
6 with 23	23	91	1363	1605	25
7 with 24	24	2400	3600	1680	26
6 with 24	24	2400	3672	1605	27
7 with 5	5	241	1441	1680	28
6 with 4	4	1431	2703	1605	29
7 with 9	9	597	1797	1680	30

Col PCR map of basic parts:

Layout of ColPCR:

Plasmid	Part number	Part Size	Eco/Xho Frags	Eco/Xho Frags	Lane #
5 with Bca1091	Bca1091	60	1260	1473	1
6 with Bjh2245	Bjh2245	97	1369	1605	2
6 with 12	12	597	1869	1605	3
5 with 25	25	41	1241	1473	4
9 with 11	11	46	1112	1605	5
9 with 23	23	91	1157	1605	6

Christoph Neyer 20:41, 23 June 2010 (EDT)

Restriction map of methylated parts Plate B:
Part 1:

Part 2:

Layout:

'	RE mapping of ALL methylated parts, Plate B	RE mapping plate layout:	'	'	'
Ladder
4 with 16	16	90	1156	1679	1
5 with 20	20	823	2023	1473	2
4 with 17	17	1845	2911	1679	3
5 with 23	23	91	1291	1473	4
4 with Bjh1882	Bjh1882	681	1747	1679	5
5 with 25	25	41	1241	1473	6
9 with 11	11	46	1112	1605	7
5 with 3	3	33	1233	1473	8
9 with 13	13	399	1465	1605	9
5 with 8	8	1244	2444	1473	10
9 with 20	20	823	1889	1605	11
5 with Bca1091	Bca1091	60	1260	1473	12
9 with 23	23	91	1157	1605	13
8 with 17	17	1845	3117	1473	14
9 with 8	8	1244	2310	1605	15
8 with 18	18	1818	3090	1473	16
Ladder
6 with 12	12	597	1869	1605	17
7 with 13	13	399	1599	1680	18
6 with 13	13	399	1671	1605	19
7 with 14	14	399	1599	1680	20
6 with 22	22	1026	2298	1605	21
7 with 19	19	228	1428	1680	22
6 with 23	23	91	1363	1605	23
7 with 2	2	35	1235	1680	24
6 with 7	7	1788	3060	1605	25
7 with 23	23	91	1291	1680	26
7 with 10	10	100	1300	1680	27
7 with 24	24	2400	3600	1680	28
7 with 11	11	46	1246	1680	29
7 with 5	5	241	1441	1680	30
8 with 4	4	1431	2703	1473	31
7 with 9	9	597	1797	1680	32
Ladder
6 with 11	11	46	1318	1605	33
5 with 15	15	90	1290	1473	34
6 with 12	12	597	1869	1605	35
5 with 3	3	33	1233	1473	36
6 with 19	19	228	1500	1605	37
8 with 1	1	35	1307	1473	38
6 with 2	2	35	1307	1605	39
8 with 21	21	232	1504	1473	40
6 with 22	22	1026	2298	1605	41
8 with 6	6	319	1591	1473	42
6 with 23	23	91	1363	1605	43
6 with 5	5	241	1513	1605	44
6 with 24	24	2400	3672	1605	45
6 with 7	7	1788	3060	1605	46
6 with 4	4	1431	2703	1605	47
6 with Bjh2245	15	90	1362	1605	48
Ladder
Gel #2
Ladder
4 with 10	10	100	1166	1679	1
4 with 18	3	33	1099	1679	2
4 with 9	18	1818	2884	1679	3
9 with 3	1	35	1101	1605	4
6 with 24	24	2400	3672	1605	5
7 with 23	23	91	1291	1680	6
7 with 24	24	2400	3600	1680	7
9 with 11	11	46	1112	1605	8
9 with 23	23	91	1157	1605	9
5 with Bca1091	Bca1091	60	1260	1473	10
4 with Bjh1882	Bjh1882	681	1747	1679	11
6 with Bjh2245	Bjh2245	97	1369	1605	12
6 with 12	12	597	1869	1605	13
5 with 25	25	41	1241	1473	14

Gel #2:

Christoph Neyer 17:44, 23 June 2010 (EDT)

Restriction map of unmethylated parts:

Christoph Neyer 13:06, 23 June 2010 (EDT)

Restriction Map of methylated parts plate rows A and E:

Layout:

'	Part number	Part Size	Eco/Xho Frags	Eco/Xho Frags	Lane #
4 with 16	16	90	1066	1769	1
9 with 13	13	399	1066	2004	2
5 with 20	20	823	1200	2296	3
5 with 8	8	1244	1200	2717	4
6 with 12	12	597	1272	2202	5
6 with 7	7	1788	1272	3393	6
7 with 13	13	399	1200	2079	7
7 with 23	23	91	1200	1771	8
6 with 11	11	46	1272	1651	9
6 with 22	22	1026	1272	2631	10
5 with 15	15	90	1200	1563	11
8 with 6	6	319	1272	1792	12
4 with 10	10	100	1066	1779	13

Christoph Neyer 13:16, 22 June 2010 (EDT)

Restriction Map Rerun:

Layout:

Restriction Mapping	'	Lane #s A	B	Part size	eco/xho frags	eco/xho frags
9 with iGEM10_023	R	1	2	1879	2945	1605
9 with iGEM10_024	R	3	4	1522	2588	1605
9 with iGEM10_028	L	5	6	157	1223	1605
9 with iGEM10_025	R	7	8	323	1389	1605
9 with iGEM10_027	L	9	10	858	1924	1605
8 with iGEM10_029	L	11	12	1909	3181	1473
9 with iGEM10_034	R2	13	14	1064	2130	1605
5 with iGEM10_035	R	15	16	996	2196	1473
7 with iGEM10_040	R	17	18	771	1971	1680
9 with iGEM10_008	R	19	20	1057	2123	1605
6 with iGEM10_033	L	21	22	1464	2736	1605

Daniela Mehech 14:19, 21 June 2010 (EDT)

Colony PCR results from Group 2

lane 1,3,5,7 = 5 w/ 35 L
lane 2,4,6,8 = 5 w/ 38
lane 9,11,13,15 = 5 w/ 35 R
lane 10,12,14,16 = 6 w/33 L
lane 17,19,21,23 = 6 w/33 R
lane 18,20,22,24 = 9 w/ 8 L
lane 25, 27,29,31 = 7 w/40
lane 26,28,30,32 = 9 w/ 34 L
lane 33,34,35,36 = 9 w/ 8 R
lane 37,38,39,40 = 9 w/ 34 R
Except for first 8 lanes, everything looked good and we mini-prepped two of each. Mini-prepped lanes 1-8 too and will send them for sequencing

Colony PCR results for group 3:

Loaded as: 1,9,2,10,3,11,4,12,5,13,6,14,7,15,8,16,17,25,18,26,19,27,20,28,21,29,22,30,23,31,24,32

Colony PCR results for group 4:

Restriction Mapping of everything we've mini-prepped (groups 1 and 2)

Right Half

Left Half

To Do

Check for co-transformation for Groups 3 and 4
Mini-Prep good colonies of group 3 and 4
Send to sequencing some parts?
Begin Stage 2 (hopefully all our stage 1 parts are right)

Daniela Mehech 21:28, 18 June 2010 (EDT)

We labeled our transformation plate wrong so when we went to plate the cells many of them were plated on the wrong antibiotics. We still had colonies grow because they were co-transformed and had resistance to all antibiotics. This also explains why so many colonies grew when we tested for co-transformation. We have split our stage 1 parts into 4 groups:

1. Colonies grew on proper antibiotics and were not co-transformed. There are 12 of these. We mini-prepped these today and will do restriction mapping properly on Monday
2. Colonies that grew on wrong antibiotics need to be redone. There are 10 of these.We digested, ligated and transformed them today.
3. Parts that we thought we hadn't done (see yesterday's notes), but it turns out we did do them without realizing it since our plates were labeled wrong. Since we did them twice and they were all plated correctly the second time we will pick colonies, do Colony PCR, check for co-transformation and mini-prep these 9 on Monday.
4. Colonies that grew on the right antibiotics but were co-transformed. We will pick more colonies from the original plate to do ColPCR

Daniela Mehech 18:37, 17 June 2010 (EDT)

We got cell growth for every part we plated (although in some parts there were very few colonies, about 3 or 4).
We made a Colony PCR Mastermix and tried to distribute it to two PCR plates with the robot but the robot kept giving us an error message (It said it couldn't pipette 19uL if there was 17uL in the tube but there was 2 ml in the tube and we wanted it to pipette 17uL). We gave up and I pipetted all 136 wells by hand.
Christoph and I picked colonies, added them to the PCR plate, innoculated them in a 96-well plate and left them in the incubator. We ran a ColPCR3K program on the both PCR plates.
Christoph noticed that we had based all of our csv robot files from the wrong list. We made all of the parts we needed but some of the parts had to be inserted into multiple vector backbones. We began making the 6 forgotten parts by hand. 3 of these parts need to be transformed into both Righties and Lefties so we're making 9 parts by hand.
We dabbed broth from our 96-well inoculated plate on both AKC plates and LB-only plates as negative and positive controls respectively for whether we picked any co-transformed colonies.
We need to run a huge gel when the PCR finishes at 4:40pm. Then we need to analyze the results. Christoph already named all the new composite parts and calculated their sizes.

To Do

We will mini-prep our good colonies tomorrow. Hopefully we'll have at least two good copies for each part so that we can continue with Plates A and B for stage 2.
We can start planning for Stage 2 assembly when we have free time. If we do it beforehand hopefully we'll catch any mistakes before running the robot
We should make a list of all the information Clothos gives us after it solves for the assembly tree. We've had to spent a good chunk of time writing excel formulas and making spreadsheets just so we know exactly how to assemble everything

Christoph Neyer 21:26, 18 June 2010 (EDT)

Did Eco/Bam digest of good parts and here is the layout on the gel:
After about 20mins:

'	'	'	'	'	Lane #	'
		part size +200	Eco/Bam frags	Eco/Bam frags	A	B
7 with iGEM10_022	R	2078	4755	4755	1	2
9 with iGEM10_023	R	2079	4624	4624	3	4
9 with iGEM10_024	R	1722	4267	4267	5	6
9 with iGEM10_028	L	357	157	2745	7	8
4 with iGEM10_036	R	282	2827	2827	9	10
4 with iGEM10_037	R	566	3111	3111	11	12
4 with iGEM10_009	R	479	3024	3024	13	14
9 with iGEM10_025	R	523	3068	3068	15	16
9 with iGEM10_027	L	1058	858	2745	17	18
8 with iGEM10_029	L	2109	1909	2671	19	20
9 with iGEM10_026	L	1535	1335	2745	21	22
9 with iGEM10_027	R	1058	3603	3603	23	24

Christoph Neyer 15:51, 18 June 2010 (EDT)

Found out that we mixed up some things while creating the plate layouts for stage1 and we ended up plating on the wrong antibiotics.

• We need to spot check and colony PCR the catchups we started yesterday. (9 total):

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_026	L	9 with 8	7 with 23
5 with iGEM10_027	L	9 with 20	7 with 2
5 with iGEM10_032	L	9 with 23	7 with 13
5 with iGEM10_034	L	9 with 20	7 with 5
5 with iGEM10_008	L	9 with 20	7 with 19
8 with iGEM10_035	L	6 with 13	4 with 9
5 with iGEM10_034	R	9 with 20	7 with 5
5 with iGEM10_008	R	9 with 20	7 with 19
8 with iGEM10_035	R	6 with 13	4 with 9

• Assembled these parts that we missed/plated on the wrong antibiotics manually:
  

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_035	L	9 with 13	7 with 9
6 with iGEM10_033	L	8 with 4	9 with 3
9 with iGEM10_034	L	5 with 20	6 with 5
9 with iGEM10_008	L	5 with 20	6 with 19
5 with iGEM10_035	L	9 with 13	7 with 9
5 with iGEM10_038	R	9 with 11	7 with 24
7 with iGEM10_040	R	4 with Bjh1882	5 with 15
6 with iGEM10_033	R	8 with 4	9 with 3
9 with iGEM10_034	R	5 with 20	6 with 5
9 with iGEM10_008	R	5 with 20	6 with 19
5 with iGEM10_035	R	9 with 13	7 with 9

Christoph Neyer 13:30, 18 June 2010 (EDT)

We spot checked our colony PCR colonies on ACK to check for co-transformation and this is what our plates look like:
Plate A

and Plate B

Christoph Neyer 21:22, 17 June 2010 (EDT)

Colony PCR results for Stage1 parts:
Here are columns 1-6 in two parts:
Part 1 (Parts 1 and 2 are picutres of the same gels)

and Part 2

Here are columns 7-10:

Christoph Neyer 18:24, 17 June 2010 (EDT)

Most of the colonies turned out good for Stage 1 of the robot assembly. We picked colonies for colony PCR.

Manually made these parts for Stage 1 using the robot protocol. We missed them some how:

Name	Strain	Lefty Input	Righty Input
5 with iGEM10_026	L	9 with 8	7 with 23
5 with iGEM10_027	L	9 with 20	7 with 2
5 with iGEM10_032	L	9 with 23	7 with 13
5 with iGEM10_034	L	9 with 20	7 with 5
5 with iGEM10_008	L	9 with 20	7 with 19
8 with iGEM10_035	L	6 with 13	4 with 9
5 with iGEM10_034	R	9 with 20	7 with 5
5 with iGEM10_008	R	9 with 20	7 with 19
8 with iGEM10_035	R	6 with 13	4 with 9

Christoph Neyer 21:15, 16 June 2010 (EDT)

While plating Stage1 2ab assembly set A we may have reversed the order of Col 1 B (30 ul).
The cells from plate A3-D3 may be in wells B4-B1 instead of B1-B4 on the 2x12 plated for Col 1A from the reaction plate.
Also note rows A and B were switched on the plate for Col 5A as noted on the plate. A is 30ul and B is 60ul instead of the other way around.

Christoph Neyer 18:10, 16 June 2010 (EDT)

Redoing Stage1B and doing Stage1A 2ab assembly today.
Check autoclave at 4:30.
Decided to redo all of B along with A. We can do them together on the robot by doubling everything but changing the source plate Made dilution plate A, and added 60uL of dilution to dilution plate B. 7 with 11 still looked different Distributed digestion cocktail to both dilution plates Note: Robot begins pipetting air if it uses the same tip for two long. We stopped the program halfway through to change its tip Distributed Righty and Lefty parts to reaction plates. Made sure there were duplicate columns for parts being transformed twice Checked that at this point all wells had the same volume Put plates in thermocycler to incubate and heat kill Distributed antibiotics to 10 plates. All of LB agar was contaminated so will pour that during the rescue step added ligation mixture to each well and let incubate at room temperature.

Christoph Neyer 18:11, 16 June 2010 (EDT)

Robot 2AB assembly Stage 1

Yesterday we did stage 1 of our 2AB assembly only on plate B. Today we will repeat the process on plate A without making all of the mistakes that we did yesterday.

June 15:
Autoclaved our strips for plating. Learned how to work the autoclave machine

Made a new plate layout for the reaction plate. Wells are organized by what strain the part needs to be transformed into to
MISTAKE: Did not listen to Tim and organized wells by column instead of rows. Rows are better because the plating strips have 2 rows and 12 columns
MISTAKE: Did not read procedure all the way through in detail and assumed that we could use a well for two transformations. Our dilution and reaction plate needs to have duplicates of the parts that go into both Righty and Lefty strains. We have to add the duplicates in either the dilution or reaction plate
Made the dilution plate B (30ul of DNA into each well + 30ul of NEB2 stock diluted by a factor of 5). Dilution plate had the same layout as methylated stock plate so we did it by hand. Next time, we should consider adding duplicates of the parts being transformed twice.
NOTE: 7 with 11 was frozen in our methylated stocks plate. This is the part that was mini-prepped separately the day before. All other wells weren't frozen.

Had the robot make reaction plate (distribute digestion mastermix to all wells, distribute all Lefty parts, mix, distribute all righty parts, mix)
While the reaction plate incubated in the black thermocycler (which also did heat kill of the digestion enzymes) we distributed the antibiotics to the 24-well strips. We made antibiotics (dilute stock by 10, add 1ul of dye). This part went fine except we should have labeled the plates before we put them on the robot. Added LB agar to the plates and let them dry next to the flame.
MISTAKE: forgot to make second plate for parts being transformed twice. Column 5 in reaction plate needed to go to two different plates. We made the extra plate by hand

Had robot add the ligation cocktail to the wells and let it incubate at room temperature. We began thawing our bacteria.
Did transformation of bacteria. We had enough volume to seed at least 60uL of bacteria per column.
MISTAKE: Did not realize that the protocol was for seeding 10uL of bacteria. We wanted to seed about 150uL. We should not have done the centrifugation and aspiration of the supernatant step.
MISTAKE: ejecting tips from multi-channel before releasing cells on plate. We lost 60uL of our cells from column 7.
MISTAKE: We split the volume in column 5 into column 9 (because column 5 had to be transformed into both righty and lefty strains). Thus we also did not have enough volume for those parts.
MISTAKE: not taking a short break after lunch to re-energize and prevent making silly mistakes

Christoph Neyer 20:10, 15 June 2010 (EDT)

• Reorganize destination plate based on L and R not on antibiotics. CHECK
• AUTOCLAVE 24 well Strips! CHECK
• Digesting (1hr) Check
  • Dilute NEB2 and DNA.
• Make plates (2hr) Check
  • Cherry pick antibiotics Check
• Heat kill (20 mins)Check
• Ligation (30 mins)Check
• Transform + Rescuing (1hr) Check
• Plate (1hr)Check

Forgot to make two copies of the parts that are both lefty and righty. So, we just used half of ligation product of each.
Here is the layout of the transformation plate:

Column 5 is Lefty and was transformed with only half the ligation product.
Column 9 is righty and was also transformed with half the ligation product.

Christoph Neyer 21:29, 14 June 2010 (EDT)

Goal for tomorrow is to transform and plate stage 1A and 1B. That involves:

• Reorganize destination plate based on L and R not on antibiotics.
• AUTOCLAVE 24 well Strips!
• Digesting (1hr)
  • Dilute NEB2 and DNA.
• Make plates (2hr)
  • Cherry pick antibiotics
• Heat kill (20 mins)
• Ligation (30 mins)
• Transform + Rescuing (1hr)
• Plate (1hr)

Christoph Neyer 14:18, 14 June 2010 (EDT)

Thanks to Tim and Shelly for picking colonies from methylation transformation

• May have mislabeled these tubes, so we are running a BglII/Xho map:

Put in digest at 11am. Run gel at 11:30.

Looks ok. Added these parts to methylated parts plates

Lane #	ID	Name	Expected fragments	'
1	4 with 10 #1	pMLL4-AC+B10sbb43	850	1990
2	4 with 10 #2	pMLL4-AC+B10sbb43	850	1990
3	4 with 18 #1	pMLL4-AC+B10sbb13	850	2160
4	4 with 18 #2	pMLL4-AC+B10sbb13	850	2160
5	4 with 9 #1	pMLL4-AC+B10Sbb20	850	2450
6	4 with 9 #2	pMLL4-AC+B10Sbb20	850	2450
7	9 with 3 #1	pMLL9-CA+Bjh1853	300	1500
8	9 with 3 #2	pMLL9-CA+Bjh1853	300	1500

Christoph Neyer 21:36, 11 June 2010 (EDT)

Plated Transformations from robot dilution.

Christoph Neyer 19:04, 11 June 2010 (EDT)

Next step is to transform each part into a Righty or Lefty strain.
Manually transformed parts from dilution plate into Righty and Lefty strains. Incubated at 5:10. Take out at 5:50.
Transformation plate has same layout as dilution plate below. Here is layout again:

Transformation plate layout

Columns 1-6 are in Righty strains. Columns 7-12 are in Lefty strains.

'

1

2

3

4

5

6

7

8

9

10

11

12

A

4 with 16

5 with 20

6 with 12

7 with 10

8 with 17

9 with 11

4 with 10

6 with 11

6 with 5

7 with 13

8 with 1

9 with 3

B

4 with 17

5 with 23

6 with 13

7 with 11

8 with 18

9 with 13

4 with 18

6 with 12

6 with 7

7 with 14

8 with 21

C

4 with Bjh1882

5 with 25

6 with 22

8 with 4

9 with 20

4 with 9

6 with 19

6 with Bjh2245

7 with 19

8 with 6

D

5 with 3

6 with 23

9 with 23

6 with 2

7 with 2

E

5 with 8

6 with 7

9 with 8

6 with 22

7 with 23

F

6 with Bca1091

6 with 23

7 with 24

G

5 with 15

6 with 24

7 with 5

H

5 with 3

6 with 4

7 with 9

Christoph Neyer 16:31, 11 June 2010 (EDT)

Did a test run with the robot using our dilution csv file. Checked cloumn 4 it worked well.
Miniprepped pMLL5+Bca1152 and sent in for sequencing as iGem021.
Using robot to dilute 4ul of each vector+part stage 0 assembly into 76ul of water. See this csv file: Media:Stage0PartsDilution.csv

Source plate layout:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

5+25 (D)

Destination plate layout:

Columns 1-6 need to be transformed into Righty strains. Columns 7-12 need to be transformed into Lefty strains.

'

1

2

3

4

5

6

7

8

9

10

11

12

A

4 with 16

5 with 20

6 with 12

7 with 10

8 with 17

9 with 11

4 with 10

6 with 11

6 with 5

7 with 13

8 with 1

9 with 3

B

4 with 17

5 with 23

6 with 13

7 with 11

8 with 18

9 with 13

4 with 18

6 with 12

6 with 7

7 with 14

8 with 21

C

4 with Bjh1882

5 with 25

6 with 22

8 with 4

9 with 20

4 with 9

6 with 19

6 with Bjh2245

7 with 19

8 with 6

D

5 with 3

6 with 23

9 with 23

6 with 2

7 with 2

E

5 with 8

6 with 7

9 with 8

6 with 22

7 with 23

F

6 with Bca1091

6 with 23

7 with 24

G

5 with 15

6 with 24

7 with 5

H

5 with 3

6 with 4

7 with 9

Christoph Neyer 13:57, 11 June 2010 (EDT)

• Added grown up Lefty and Righty strains to 500ml each of LB. Growing up for 2-3hrs. Check at 1pm.
• Running colony PCR gel for Bca1152+pMLL5. Expected band at ~260.

Colonies A,C,D look good. Chose C and D to miniprep. Cells were put in warm room at 9am.
Grab Bca1152 colony PCR colonies at 1 or 2pm.

• Added missing parts to iGEM10 plate1 minis:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

Lysis Device

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

5+25

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

Christoph Neyer 21:03, 10 June 2010 (EDT)

TO DO:

• Pick colonies from Bca1152 and do colony PCR. Then miniprep if time.
• Play with robot
• Make competent cells.
• Transfer box parts to dilutions plate.
• When oligos arrive. Attempt biobrick on Bjh2342CA.

Christoph Neyer 13:38, 10 June 2010 (EDT)

• Had to re-grow Lefty and Righty strains since no colonies grew.
• Need to make part 5 with 25 (pMLL5-CK+B10sbb37) or (pMLL5-CK+Bca1152)
  

Currently in DH10B. Need to Eco/Bam transfer it into pMLL5-CK.
Put E/B digest of Bca1152 in DH10B into thermocycler at 12:10pm. Take out at 1:10pm and do small fragment zymo cleanup.
Set up ligation of Bca1152 and pMLL5 at 2:15. Transform at 2:45.
Heat-shock transformed Bca1152+pMLL5 into Jtk049. Plate at 4:15pm.
Plated Bca1152+pMLL5 into Jtk049. Growing up over night.
Sent in iGEM020 for sequencing(6 with 12C from working box to double check).

Christoph Neyer 20:31, 9 June 2010 (EDT)

TO DO List:

• Finish generating competent cells for "Lefty" and "Righty" strains.
• Analyze sequencing data for pMLL6+Bjh2245 B and C.
• Transform pMLL6+Bjh2245 into "Righty" strain.
• Transform other parts into the correct "Righty" or "Lefty" strains to prepare for robot 2ab assembly.

Christoph Neyer 15:16, 9 June 2010 (EDT)

Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good. Wait for colonies to grow and then miniprep.

• Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing.
• Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
• Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
• Miniprepped pMLL6+Bjh2245. Need to transform into "Righty strain"
• Sent in minprepped pMLL6+Bjh2245 to be sequenced.

Christoph Neyer 13:28, 9 June 2010 (EDT)

Bca1091 is 60bps.
Colony PCR band for parts:

• pMLL4+bjh1882 = 849bps
• pMLL6+bjh2245 = 265bps
• pMLL5+bca1091 = 228bps

• For First try Colony PCR of 1882 and 1091:
  • Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)

• For Second try Colony PCR of 1882 and 1091
  • Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)

Christoph Neyer 19:37, 8 June 2010 (EDT)

TO DO Tomorrow:

• Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot.
• Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly.

Christoph Neyer 19:51, 8 June 2010 (EDT)

• Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091 AGAIN:

ladder

1882a

b

c

d

1901a(1)

b

c

d

ladder

1901a(2)

b

c

d

Christoph Neyer 16:59, 8 June 2010 (EDT)

• Ligated pMLL6 Eco/Bam digest with part Bjh2245.
• Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:

ladder

1882a

b

c

d

1901a(1)

b

c

d

ladder

1901a(2)

b

c

d

• Repicking colonies since Colony PCR appeared contaminated. Block has same layout, shown below:

PCR Block 1882 and 1091 #2	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D

• Ran out of "Righty" cells. So we are transforming Jtk049 with our E/B ligation of pMLL6+Bjh2245.

Christoph Neyer 14:04, 8 June 2010 (EDT)

• Digested pMLL6 w/ Eco/Bam and gel purified.
• Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
  

PCR Block 1882 and 1091 #1	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D

Christoph Neyer 19:30, 7 June 2010 (EDT)

Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:

• Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
  
• Check transformed Bjh1882 and Bca1091.
• Transform ligated Bjh2245+pMLL6 into Righty strain

Christoph Neyer 16:35, 7 June 2010 (EDT)

Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.


Gel purified Bjh1882.

Christoph Neyer 15:54, 7 June 2010 (EDT)

Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.