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Revision as of 22:27, 3 October 2013 (view source) TaniaChristiansen (Talk | contribs) (Created page with " ==Amplifications== ===Amplification of fragment 1=== ====A==== PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5 ...") ← Older edit		Revision as of 00:05, 4 October 2013 (view source) TaniaChristiansen (Talk | contribs) Newer edit →
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	====D====		====D====
-	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.JPG|thumbnail|right|PCR Results	+	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results
	of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and		of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and
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	| 1  || 12 || inf		| 1  || 12 || inf
	|}		|}
		+
	====E====		====E====
	'''Amplification with Phusion Flash'''		'''Amplification with Phusion Flash'''
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	|}		|}
	====B====		====B====
-	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.JPG|thumbnail|right|PCR Results	+	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results
	of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and		of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and
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	|}		|}
	====B====		====B====
-	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.JPG|thumbnail|right|PCR Results	+	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results
	of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and		of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and
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	|}		|}
	====B====		====B====
-	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.JPG|thumbnail|right|PCR Results	+	[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results
	of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and		of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and

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Revision as of 00:05, 4 October 2013

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	--