Team:Heidelberg/Templates/Del week13 AE

From 2013.igem.org

(Difference between revisions)
(Created page with "==26-07-2013== ===Restriction digest of fragment FS_02 to FS_03; 5.3 kb; 08-07-2013 with EcoRI-HF=== [[File:20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII O...")
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==26-07-2013==
==26-07-2013==
===Restriction digest of fragment FS_02 to FS_03; 5.3 kb; [[DelA-E#08-07-2013|08-07-2013]] with EcoRI-HF===
===Restriction digest of fragment FS_02 to FS_03; 5.3 kb; [[DelA-E#08-07-2013|08-07-2013]] with EcoRI-HF===
-
[[File:20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII OPEcoRIHFbesch.png|100px|thumb|Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII OPEcoRIHFbesch.png|100px|thumb|Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)]]
Incubation at 37°C for 1 h 45 min
Incubation at 37°C for 1 h 45 min
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==27-07-2013==
==27-07-2013==
===Amplificaction from FS_02 to FS_03; 5.3 kb===
===Amplificaction from FS_02 to FS_03; 5.3 kb===
-
[[File:20130727 2log 2xAE 2xAF 1kb 2xIliabesch.png|100px|thumb|Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130727 2log 2xAE 2xAF 1kb 2xIliabesch.png|100px|thumb|Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)]]
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[[File:20130727 2log 2xAE 2xAF 1kb 2xIliacut.png|100px|thumb|Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130727 2log 2xAE 2xAF 1kb 2xIliacut.png|100px|thumb|Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)]]
:'''Reaction'''
:'''Reaction'''
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! what !! µL   
! what !! µL   
|-
|-
-
| ''D. acidovorans'' || 1
+
| ''D. acidovorans'' DSM-39 || 1
|-
|-
| FS_02:  (1/10) || 2.5
| FS_02:  (1/10) || 2.5
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===Amplificaction from FS_02 to FS_03; 5.3 kb===
===Amplificaction from FS_02 to FS_03; 5.3 kb===
-
[[File:20130728 1kbladder 2x DelAE FS2to3 5u prim 2x DelAE FS2to3 2,5u prim.png|100px|thumb|2nd Amplification of DelAE (FS02 to FS03; 27.07,1), first two lanes with 5 µL primer, second two lanes with 2.5 µL primer; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130728 1kbladder 2x DelAE FS2to3 5u prim 2x DelAE FS2to3 2,5u prim.png|100px|thumb|2nd Amplification of DelAE (FS02 to FS03; 27.07,1), first two lanes with 5 µL primer, second two lanes with 2.5 µL primer; run at 100 V, 0.8 % gel (TAE)]]
-
[[File:20130728 1kbladder 2x DelAE FS2to3 5u prim 2x DelAE FS2to3 2,5u prim cut.png|100px|thumb| gel after excision]]
+
[[File:Heidelberg_20130728 1kbladder 2x DelAE FS2to3 5u prim 2x DelAE FS2to3 2,5u prim cut.png|100px|thumb| gel after excision]]
   
   
:'''Reaction'''
:'''Reaction'''
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! what !! µL   
! what !! µL   
|-
|-
-
| ''D. acidovorans'' || 1.5/1
+
| ''D. acidovorans'' DSM-39 || 1.5/1
|-
|-
| FS_02:  (1/10) || 2.5/5
| FS_02:  (1/10) || 2.5/5
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===Amplification I from FS_02 to FS_24; 7.1 kb===
===Amplification I from FS_02 to FS_24; 7.1 kb===
-
[[File:20130627 log2 1kbladder FS02toFS24,200µM68td FS02toFS24,400µM68td FS02toFS24,200µM65 FS02toFS24,400µM65 FS06toFS24 FS20toFS24.png|200px|thumb| Amplifikation of DelFG (FS02-FS24), ; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130627 log2 1kbladder FS02toFS24,200µM68td FS02toFS24,400µM68td FS02toFS24,200µM65 FS02toFS24,400µM65 FS06toFS24 FS20toFS24.png|200px|thumb| Amplifikation of DelFG (FS02-FS24), ; run at 100 V, 0.8 % gel (TAE)]]
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
:'''Reaction'''
:'''Reaction'''
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! what !! µl   
! what !! µl   
|-
|-
-
| ''D. acidovorans'' || 1
+
| ''D. acidovorans'' DSM-39 || 1
|-
|-
| FS_02:  (1/10) || 4/2
| FS_02:  (1/10) || 4/2
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===Amplification II from FS_02 to FS_24; 7.1 kb===
===Amplification II from FS_02 to FS_24; 7.1 kb===
-
[[File:20130727 2log 1kb 2-24.200nM.68td 2-24.400nM.68td 2-23.200nM.68td 2-23.400nM.68td 2-23.200nM.65 2-23.400nM.65.png|150px|thumb| 2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V ]]
+
[[File:Heidelberg_20130727 2log 1kb 2-24.200nM.68td 2-24.400nM.68td 2-23.200nM.68td 2-23.400nM.68td 2-23.200nM.65 2-23.400nM.65.png|150px|thumb| 2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V ]]
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! what !! µl   
! what !! µl   
|-
|-
-
| ''D. acidovorans'' || 1
+
| ''D. acidovorans'' DSM-39 || 1
|-
|-
| FS_02:  (1/10) || 2/4
| FS_02:  (1/10) || 2/4
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===Amplification III from FS_02 to FS_24; 7.1 kb===
===Amplification III from FS_02 to FS_24; 7.1 kb===
-
[[File:20130728 1kbruler 4xFS02-FS23.60const.@T100.200nM T100.400nM old.200nM old.400nM red.200nM red.400nM FS02-FS24.janusA.200nM.66td FS02-FS24.T100.400nM.60const 1kb FS04-FS24.png|150px|thumb| 1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130728 1kbruler 4xFS02-FS23.60const.@T100.200nM T100.400nM old.200nM old.400nM red.200nM red.400nM FS02-FS24.janusA.200nM.66td FS02-FS24.T100.400nM.60const 1kb FS04-FS24.png|150px|thumb| 1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)]]
-
[[File:20130728 1kbruler 4xFS02-FS23.60const.@T100.200nM T100.400nM old.200nM old.400nM red.200nM red.400nM FS02-FS24.janusA.200nM.66td FS02-FS24.T100.400nM.60const 1kb FS04-FS24 cut.png|150px|thumb| 1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130728 1kbruler 4xFS02-FS23.60const.@T100.200nM T100.400nM old.200nM old.400nM red.200nM red.400nM FS02-FS24.janusA.200nM.66td FS02-FS24.T100.400nM.60const 1kb FS04-FS24 cut.png|150px|thumb| 1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)]]
2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers
2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers
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! what !! µl   
! what !! µl   
|-
|-
-
| ''D. acidovorans'' || 1
+
| ''D. acidovorans'' DSM-39 || 1
|-
|-
| FS_02:  (1/10) || 2/4
| FS_02:  (1/10) || 2/4
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==29-07-2013==
==29-07-2013==
===Restriction digest of FS_02 to FS_03; 5.3 kb;([[DelA-E#27-07-2013|27-07-2013]]; II) with BglII ===
===Restriction digest of FS_02 to FS_03; 5.3 kb;([[DelA-E#27-07-2013|27-07-2013]]; II) with BglII ===
-
[[File:20130729restrictiondigest 2log FS02-FS03 FS02-FS05 FS08-FS23besch.png|100px|thumb|Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)]]
+
[[File:Heidelberg_20130729restrictiondigest 2log FS02-FS03 FS02-FS05 FS08-FS23besch.png|100px|thumb|Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)]]
Incubation at 37°C for about 3 h
Incubation at 37°C for about 3 h
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|}
|}
Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb
Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb
-
[[File:AE2-3 BglII.png|60px]]
+
 
'''Results:'''
'''Results:'''
* Restriction digest shows the expected product sizes
* Restriction digest shows the expected product sizes
* indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC
* indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC

Revision as of 01:36, 30 September 2013

Contents

26-07-2013

Restriction digest of fragment FS_02 to FS_03; 5.3 kb; 08-07-2013 with EcoRI-HF

Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 1 h 45 min

what µL
FS_02 to FS_03 (08-07-2013) 15
EcorRI-HF 0.5
Buffer CutSmart 2
dd H2O 2

Expected fragment lengths: 3054 bp, 2260 bp

Results:

  • restriction digest of DelAE did not work, since incubation time might have been to short

27-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 2.5
FS_03: (1/10) 2.5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 19


Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 12 min
1 12 inf

Results:

  • Amplification of DelAE didnt work out, only smear occured
  • repeat PCR with better cycler

Amplificaction from FS_02 to FS_03; 5.3 kb

2nd Amplification of DelAE (FS02 to FS03; 27.07,1), first two lanes with 5 µL primer, second two lanes with 2.5 µL primer; run at 100 V, 0.8 % gel (TAE)
gel after excision
Reaction
what µL
D. acidovorans DSM-39 1.5/1
FS_02: (1/10) 2.5/5
FS_03: (1/10) 2.5/5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 19/14
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 12 min
1 12 inf

Results:

  • Amplification of DelAE worked with both 200 and 400 nM of Primers, nevertheless amplification was more specific with the higher primer concentration
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification I from FS_02 to FS_24; 7.1 kb

Amplifikation of DelFG (FS02-FS24), ; run at 100 V, 0.8 % gel (TAE)

4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 4/2
FS_24: (1/10) 4/2
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 3:50
1 72 12 min
1 10 inf


Conditions II
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:50 min
18 98 1
66 5
72 3:50 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAE worked with 200 nM primer concentration at an annealing temperature of 68°C and 400 nM at an annealing temperature of 65°C, the product obtained at 65°C was more specific
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification II from FS_02 to FS_24; 7.1 kb

2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V


2 reactions, 68°C Touchdown with 200nM Primers and 65°C constant with 400nM Primers

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2/4
FS_24: (1/10) 2/4
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 5:40
1 72 12 min
1 10 inf


Conditions II
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 5:40 min
18 98 1
66 5
72 5:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification did not work, neither with 200nM and 68°C touchdown, nor with 400nM and 65°C constant.
  • Repeat amplfication with different conditions as primers did not bind very effectively

Amplification III from FS_02 to FS_24; 7.1 kb

1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)
1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)

2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2/4
FS_24: (1/10) 2/4
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 5:40
1 72 12 min
1 10 inf


Conditions II
Biorad C1000 Touch Block A
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 5:40 min
18 98 1
64 5
72 5:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification from DelAE (7.1 kbp) failed again
  • stick to the old strategy and use previously obtained fragments with different other fragments for gibson assembly.

29-07-2013

Restriction digest of FS_02 to FS_03; 5.3 kb;(27-07-2013; II) with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 h

what µL
FS_02 to FS_03 (27-07-2013; II) 15
BglII 1
Buffer 3.1 2
dd H2O 2

Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb


Results:

  • Restriction digest shows the expected product sizes
  • indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week13_AE"