"

From 2013.igem.org

Contents 1 Amplifications 1.1 Amplification of fragment 1 1.1.1 A 1.1.2 B 1.2 Amplification of fragment 2 1.2.1 A 1.2.2 B 1.3 Amplification of fragment 3 1.3.1 A 1.3.2 B 1.3.3 C 1.3.4 D 1.3.5 E 1.3.6 F 1.4 Amplification of fragment 4 1.4.1 A 1.4.2 B 1.4.3 C 1.5 Amplification of fragment 5 1.5.1 A 1.5.2 B 1.6 Amplification of fragment 6 1.6.1 A 1.6.2 B 1.7 Amplification of fragment 7 1.7.1 A 1.7.2 B 1.8 Amplification of fragment 8 1.8.1 A 1.8.2 B 1.9 Amplification of fragment 9 1.9.1 A 1.9.2 B 1.10 Amplification of fragment 10 1.10.1 A 1.10.2 B 1.11 Amplification of fragment 11 1.11.1 A 1.12 Amplification of fragment 12 1.12.1 A 1.13 Amplification of fragment 13 1.13.1 A 1.13.2 B 1.14 Amplification of fragment 14 1.14.1 A 1.15 Amplification of fragment 15 1.15.1 A 1.15.2 B 2 Analysis of DNA concentrations of previously amplified fragments

Amplifications

Amplification of fragment 1

A

what	µl
pSB1C3	0,5
IK22	2
IK23	2
Q5 2x Master mix	10
ddH20	5,5

Cycles	temperature [°C]	Time [s]
1	98	120
35	98	5
	66	10
	72	120
1	72	600
1	12	inf

Result band in gel--> cut and extraction

B

what	µl
IK23	2
IK22	2
pSB1C3	0.5 (for fragments 1, 4 & 9)
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:00
1	72	10:00
1	12	inf

Amplification of fragment 2

A

what	µl
B. parabrevis	1
IK13	2
IK14	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [s]
1	98	300
35	98	5
	70	10
	72	60
1	72	600
1	12	inf

Result Successful

B

what	µl
Bacillus	1
IK13	2
IK14	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	70	0:15
	72	1:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 3

A

what	µl
B. parabrevis	1
IK12	2
IK15	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [s]
1	98	300
35	98	5
	66	10
	72	120
1	72	600
1	12	inf

Result no band; new PCR settings

B

Cycles	temperature [°C]	Time [s]
1	98	300
12	98	5
	70 touchdown (-0.5°C)	5
	72	180
23	98	5
	66	10
	72	180
1	72	600
1	12	inf

Result xxxxxxxx; new: touchdown PCR with Phusion Flash

C

what	µl
primer fw	2
primer rv	2
DMSO	1
B. parabrevis	1
Phusion flash	10
ddH20	4

Cycles	temperature [°C]	Time [s]
1	98	120
12	98	5
	70 touchdown (-0.5°C)	5
	72	120
23	98	5
	64	5
	72	120
1	72	600
1	12	inf

Result Just a light band, have to improve conditions.

D

Amplification with Q5

what	µl
Bacillus	1
IK12	2
IK15	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	2:00
1	72	10:00
1	12	inf

E

Amplification with Phusion Flash

what	µl
Bacillus	1
IK12	2
IK15	2
Phusion 2x Master mix	10
DMSO	1
ddH20	4

Cycles	temperature [°C]	Time [s]
1	98	2:00
12	98	0:05
	70↓0.5°C	0:05
	72	2:30
23	98	0:05
	64	0:05
	72	3:00
1	72	10:00
1	12	inf

F

what	µl
Bacillus	1
IK12	2
IK15	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	70	0:15
	72	2:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 4

A

what	µl
pSB1C3	0,5
IK21 (1:10)	2
IK22 (1:10)	2
Q5 2x Master mix	10
ddH20	5,5

Cycles	temperature [°C]	Time [s]
1	98	300
35	98	5
	66 (fragment 4)	10
	72	120
1	72	600
1	12	inf

Result Successful

B

what	µl
pSB1C3	0.5
IK21	2
IK22	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:00
1	72	10:00
1	12	inf

Result PCR redone over night as gel was clumpy and did not show the expected fragments.

C

what	µl
IK21	2
IK22	2
pSB1C3	0.5
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:00
1	72	10:00
1	12	inf

Amplification of fragment 5

A

what	µl
B. Parabrevis	1
IK16	2
IK17	2
Q5 2x Master mix	10
ddH20	5,0

Cycles	temperature [°C]	Time [s]
1	98	120
35	98	5
	65	10
	72	60
1	72	600
1	12	inf

B

what	µl
Bacillus	1
IK16	2
IK17	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:30
1	72	10:00
1	12	inf

Amplification of fragment 6

A

what	µl
B. Parabrevis	1
IK12	2
IK18	2
Q5 2x Master mix	10
ddH20	5,0

Cycles	temperature [°C]	Time [s]
1	98	120
35	98	5
	66	10
	72	150
1	72	600
1	12	inf

B

what	µl
Bacillus	1
IK12	2
IK18	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	3:00
1	72	10:00
1	12	inf

Amplification of fragment 7

A

what	µl
B. Parabrevis	1
IK13	2
IK19	2
Q5 2x Master mix	10
ddH20	5,0

Cycles	temperature [°C]	Time [s]
1	98	120
35	98	5
	70	10
	72	60
1	72	600
1	12	inf

B

what	µl
Bacillus	1
IK13	2
IK19	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	70	0:15
	72	1:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

Amplification of fragment 8

A

what	µl
B. Parabrevis	1
IK20	2
IK12	2
Q5 2x Master mix	10
ddH20	5,0

Cycles	temperature [°C]	Time [s]
1	98	120
35	98	5
	66	10
	72	150
1	72	600
1	12	inf

B

what	µl
Bacillus	1
IK12	2
IK20	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	3:00
1	72	10:00
1	12	inf

Amplification of fragment 9

A

what	µl
pSB1C3	0.5
PW04	2
IK21	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

what	µl
PW04	2
IK22	2
pSB1C3	0.5
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	1:00
1	72	10:00
1	12	inf

Amplification of fragment 10

A

what	µl
Bacillus	1
PW05	2
PW06	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	70	0:15
	72	1:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

what	µl
PW05	2
PW06	2
Bacillus	1
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	70 (fragment 10)	0:15
	72	1:00
1	72	10:00
1	12	inf

Amplification of fragment 11

A

what	µl
PW07	2
PW08	2
Bacillus	1
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	72	0:15
	72	2:30
1	72	10:00
1	12	inf

Amplification of fragment 12

A

what	µl
PW09	2
PW10	2
Bacillus	1
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	69	0:15
	72	1:30
1	72	10:00
1	12	inf

Amplification of fragment 13

A

what	µl
Bacillus	1 (for fragments 2, 3, 7, 10, 13 & 15)
PW11	2
IK12	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	2:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

what	µl
PW11	2
IK12	2
Bacillus	1
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	2:00
1	72	10:00
1	12	inf

Amplification of fragment 14

A

what	µl
PW09	2
IK12	2
Bacillus	1 (for fragments 2, 10, 13, 14 & 15)
pSB1C3	0.5 (for fragments 1, 4 & 9)
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	62	0:15
	72	1:00
1	72	10:00
1	12	inf

Amplification of fragment 15

A

what	µl
Bacillus	1 (for fragments 2, 3, 7, 10, 13 & 15)
PW13	2
IK12	2
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	2:00
1	72	10:00
1	12	inf

PCR redone over night as gel was clumpy and did not show the expected fragments.

B

what	µl
PW13	2
IK12	2
Bacillus	1
Q5 2x Master mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	66	0:15
	72	2:00
1	72	10:00
1	12	inf

Analysis of DNA concentrations of previously amplified fragments

Analytical Gel Electrophoresis 0.8% agarose Scheme: each 1µL DNA (eluation in 20µL water)+ 3 µL loading dye
2log ladder, 4 µL 24.7:4, 25.7:1,2,3,7, 26.7.:3,5,6,8,3', 28.7.1,2,4,9,10,13,14 2log ladder, 4 µL

fragment	concentration [ng/µl]
1	32
2	16
3	--
4	30
5	12
6	8
7	12
8	8
9	30
10	40
11	in progress
12	in progress
13	4
14	40
15	--