Team:Heidelberg/Tyrocidine week13 ms

From 2013.igem.org

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Line 4: Line 4:
===Amplification of fragment 1===
===Amplification of fragment 1===
====A====
====A====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 43: Line 43:
====B====
====B====
-
[[File:Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
+
[[File:Heidelberg_Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
  {| class="wikitable"
  {| class="wikitable"
Line 80: Line 80:
===Amplification of fragment 2===
===Amplification of fragment 2===
====A====
====A====
-
[[File:B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
+
[[File:Heidelberg_B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
Line 122: Line 122:
====B====
====B====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 163: Line 163:
===Amplification of fragment 3===
===Amplification of fragment 3===
====A====
====A====
-
[[File:B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
+
[[File:Heidelberg_B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
Line 204: Line 204:
no band; new PCR settings
no band; new PCR settings
====B====
====B====
-
[[File:20130725_4µl_log2_IK15toIK12.png|right|thumbnail|100px|Lane 1: NEB  
+
[[File:Heidelberg_20130725_4µl_log2_IK15toIK12.png|right|thumbnail|100px|Lane 1: NEB  
2-log; lane 2: ''B. parabrevis'' with fragment 3]]
2-log; lane 2: ''B. parabrevis'' with fragment 3]]
Line 234: Line 234:
xxxxxxxx; new: touchdown PCR with Phusion Flash
xxxxxxxx; new: touchdown PCR with Phusion Flash
====C====
====C====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 281: Line 281:
====D====
====D====
-
[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results  
+
[[File:Heidelberg_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  
Line 364: Line 364:
====F====
====F====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 404: Line 404:
===Amplification of fragment 4===
===Amplification of fragment 4===
====A====
====A====
-
[[File:B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
+
[[File:Heidelberg_B.brevis-PCR2013-07-24.png|right|thumb|100px| Lane 1: NEB 2-log;  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
lane 2: pJM5 with primers IK21+IK22; lane 3: ''B. parabrevis'' with primers  
Line 447: Line 447:
====B====
====B====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 488: Line 488:
====C====
====C====
-
[[File:Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
+
[[File:Heidelberg_Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
Line 526: Line 526:
===Amplification of fragment 5===
===Amplification of fragment 5===
====A====
====A====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 561: Line 561:
|}
|}
====B====
====B====
-
[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results  
+
[[File:Heidelberg_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  
Line 601: Line 601:
===Amplification of fragment 6===
===Amplification of fragment 6===
====A====
====A====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 637: Line 637:
|}
|}
====B====
====B====
-
[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results  
+
[[File:Heidelberg_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  
Line 677: Line 677:
===Amplification of fragment 7===
===Amplification of fragment 7===
====A====
====A====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 713: Line 713:
|}
|}
====B====
====B====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 754: Line 754:
===Amplification of fragment 8===
===Amplification of fragment 8===
====A====
====A====
-
[[File:20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
+
[[File:Heidelberg_20130725_4µL2log_tyr3-8.png|thumbnail|right|PCR Results of 25.07.13  
2-log ladder and Tyr 3-8 with Q5]]
2-log ladder and Tyr 3-8 with Q5]]
Line 790: Line 790:
|}
|}
====B====
====B====
-
[[File:20130726 2log TyrQ53 5 6 8 Ph 3 TC.png|thumbnail|right|PCR Results  
+
[[File:Heidelberg_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  
Line 829: Line 829:
===Amplification of fragment 9===
===Amplification of fragment 9===
====A====
====A====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 868: Line 868:
'''PCR redone over night as gel was clumpy and did not show the expected fragments.'''
'''PCR redone over night as gel was clumpy and did not show the expected fragments.'''
====B====
====B====
-
[[File:Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
+
[[File:Heidelberg_Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
  {| class="wikitable"
  {| class="wikitable"
Line 905: Line 905:
===Amplification of fragment 10===
===Amplification of fragment 10===
====A====
====A====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 979: Line 979:
===Amplification of fragment 11===
===Amplification of fragment 11===
====A====
====A====
-
[[File:20130729 2LOG Tyr11,12.png|right|thumbnail|amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing]]
+
[[File:Heidelberg_20130729 2LOG Tyr11,12.png|right|thumbnail|amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing]]
Line 1,017: Line 1,017:
===Amplification of fragment 12===
===Amplification of fragment 12===
====A====
====A====
-
[[File:20130729 2LOG Tyr11,12.png|right|thumbnail|amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing]]
+
[[File:Heidelberg_20130729 2LOG Tyr11,12.png|right|thumbnail|amplification of fragments 11 & 12. Fragment 11 is clearly visible, fragment 12 is missing]]
Line 1,055: Line 1,055:
===Amplification of fragment 13===
===Amplification of fragment 13===
====A====
====A====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 1,093: Line 1,093:
'''PCR redone over night as gel was clumpy and did not show the expected fragments.'''
'''PCR redone over night as gel was clumpy and did not show the expected fragments.'''
====B====
====B====
-
[[File:Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
+
[[File:Heidelberg_Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
  {| class="wikitable"
  {| class="wikitable"
|-
|-
Line 1,129: Line 1,129:
===Amplification of fragment 14===
===Amplification of fragment 14===
====A====
====A====
-
[[File:Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
+
[[File:Heidelberg_Tyrocidine 1,4,9,10,13,14,15.png|right|thumbnail|all constructs extracted by gel extraction; fragment 15 missing -> Optimization]]
  {| class="wikitable"
  {| class="wikitable"
Line 1,168: Line 1,168:
===Amplification of fragment 15===
===Amplification of fragment 15===
====A====
====A====
-
[[File:Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
+
[[File:Heidelberg_Tyrocidine 2,3,4,7,9,10,13,15.png|thumbnail|right|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]]
Line 1,244: Line 1,244:
''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''<br>
''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''<br>
-
[[File:Tyrocidine quantification gel.png|400px|right|thumbnail|quantification gel of the fragments extracted from gel. See table for precise amounts]]
+
[[File:Heidelberg_Tyrocidine quantification gel.png|400px|right|thumbnail|quantification gel of the fragments extracted from gel. See table for precise amounts]]
{| class="wikitable"
{| class="wikitable"

Latest revision as of 16:12, 4 October 2013

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"