Team:Heidelberg/Tyrocidine week13 ms

From 2013.igem.org

Contents

Amplifications

Amplification of fragment 1

A

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

all constructs extracted by gel extraction; fragment 15 missing -> Optimization
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

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12
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

Lane 1: NEB 2-log; lane 2: B. parabrevis with fragment 3
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

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit

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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

Lane 1: NEB 2-log; lane 2: pJM5 with primers IK21+IK22; lane 3: B. parabrevis with primers IK13+IK14; lane 4: B. parabrevis with primers IK15+IK12
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

all constructs extracted by gel extraction; fragment 15 missing -> Optimization


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

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit
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

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit
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

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

PCR Results of 25.07.13 2-log ladder and Tyr 3-8 with Q5
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

PCR Results of 26.07.13 Tyr 3,5,6,8 with Q5 and 3' with Phusion Flash. All were cut and extracted with Qiagen Gel Extraction Kit
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

all constructs extracted by gel extraction; fragment 15 missing -> Optimization
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing


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

amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing


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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

all constructs extracted by gel extraction; fragment 15 missing -> Optimization
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

all constructs extracted by gel extraction; fragment 15 missing -> Optimization
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

PCR Results of 27.07.2013 PCRs of fragments 2, 3, 4, 7, 9, 10, 13 & 15, fragment 2 and 7 were cut out for gel extraction


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

quantification gel of the fragments extracted from gel. See table for precise amounts
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 --
Retrieved from "http://2013.igem.org/Team:Heidelberg/Tyrocidine_week13_ms"