Team:Heidelberg/Templates/DelH week11
From 2013.igem.org
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[[File:Heidelberg_20130708 Del1b M Del1b+DMSO.png|200px|thumb|right|'''Fig.11.1''' gel of amplified fragment F1B(loaded 20 µL) <br> ''l1:''F1b without DMSO, ''l2:'' 2log ladder, ''l3:'' F1b with DMSO ]] | [[File:Heidelberg_20130708 Del1b M Del1b+DMSO.png|200px|thumb|right|'''Fig.11.1''' gel of amplified fragment F1B(loaded 20 µL) <br> ''l1:''F1b without DMSO, ''l2:'' 2log ladder, ''l3:'' F1b with DMSO ]] | ||
- | + | ||
There is a specific band, but not at 5 Kb. | There is a specific band, but not at 5 Kb. | ||
:=> We cannot amplify F1b from a PCR fragment. Use fresh bacteria instead. | :=> We cannot amplify F1b from a PCR fragment. Use fresh bacteria instead. | ||
<br/> | <br/> | ||
+ | <div style="clear:both"></div> | ||
====Preparation of fresh ACM media==== | ====Preparation of fresh ACM media==== | ||
* Mix ingrediants according to [https://2013.igem.org/Team:Heidelberg/Materials Acidovorax complex medium] | * Mix ingrediants according to [https://2013.igem.org/Team:Heidelberg/Materials Acidovorax complex medium] | ||
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[[File:Heidelberg_201307112log F1b-DMSO F1b+DMSO.png|200px|thumb|right|'''Fig.11.2''' gel of amplified fragment 1B(loaded 20 µL) <br> ''l1:''F1b without DMSO, ''l2:'' 2log ladder, ''l3:'' F 1b with DMSO <br/> ''l3'' shows a slight band at 5 Kb]] | [[File:Heidelberg_201307112log F1b-DMSO F1b+DMSO.png|200px|thumb|right|'''Fig.11.2''' gel of amplified fragment 1B(loaded 20 µL) <br> ''l1:''F1b without DMSO, ''l2:'' 2log ladder, ''l3:'' F 1b with DMSO <br/> ''l3'' shows a slight band at 5 Kb]] | ||
- | + | ||
<br/> | <br/> | ||
There is a weak band at the probe 1b WITH DMSO above 5 Kb. | There is a weak band at the probe 1b WITH DMSO above 5 Kb. | ||
: => Start a new PCR with 50 µl with the same conditions. | : => Start a new PCR with 50 µl with the same conditions. | ||
<br/> | <br/> | ||
+ | <div style="clear:both"></div> | ||
====PCR Conditions F1b.W11.C==== | ====PCR Conditions F1b.W11.C==== | ||
{| class="wikitable" style="float:left; margin-right:1em" | {| class="wikitable" style="float:left; margin-right:1em" | ||
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Expected band: 5 Kb. | Expected band: 5 Kb. | ||
<br/> | <br/> | ||
- | |||
[[File:Heidelberg_20130711 2log 3x1b cut.png|200px|thumb|right|'''Fig.11.4'''gel of amplified fragment 1B(loaded 50 µL) <br> ''l1:''2log ladder, ''l2-4:'' F 1b with DMSO <br/> ''l2-4'' band at 5 Kb was cut out]] | [[File:Heidelberg_20130711 2log 3x1b cut.png|200px|thumb|right|'''Fig.11.4'''gel of amplified fragment 1B(loaded 50 µL) <br> ''l1:''2log ladder, ''l2-4:'' F 1b with DMSO <br/> ''l2-4'' band at 5 Kb was cut out]] | ||
- | <div style="clear: | + | <div class="tright" style="clear:none">[[File:Heidelberg_20130711 2log 3x1b.png|200px|thumb|right|'''Fig.11.3''' gel of amplified fragment 1B (loaded 50 µL) <br> ''l1:''2log ladder, ''l2-4:'' F 1b with DMSO <br/> ''l2-4'' show expected band at 5 Kb and another lower band]]</div> |
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The expected band at 5 Bk is weak, but visible. Note that there is sample left in the pocket. | The expected band at 5 Bk is weak, but visible. Note that there is sample left in the pocket. | ||
:=> The fragment was cut and gel extracted. | :=> The fragment was cut and gel extracted. | ||
<br/> | <br/> | ||
+ | |||
+ | <div style="clear:both"></div> | ||
===Generation of DelH plasmid=== | ===Generation of DelH plasmid=== | ||
====Restriction Digest==== | ====Restriction Digest==== | ||
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[[File:Heidelberg_20130713 1a 2log 1b 2 BB.png|200px|thumb|right|'''Fig.11.5'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | [[File:Heidelberg_20130713 1a 2log 1b 2 BB.png|200px|thumb|right|'''Fig.11.5'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | ||
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No band visible. | No band visible. | ||
:=> Gel extract earlier PCR from 06-07. | :=> Gel extract earlier PCR from 06-07. | ||
<br/> | <br/> | ||
+ | <div style="clear:both"></div> | ||
Expected band: 5 Kb | Expected band: 5 Kb | ||
<br/> | <br/> | ||
[[File:Heidelberg_20130713 1a 2log miniprepBB PCRBB.png|200px|thumb|right|'''Fig.11.6'''gel of minipreped and PCR-amplified DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' Miniprep of BB (pSB6A1), ''l4:''PCR of BB (pSB6A1)]] | [[File:Heidelberg_20130713 1a 2log miniprepBB PCRBB.png|200px|thumb|right|'''Fig.11.6'''gel of minipreped and PCR-amplified DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' Miniprep of BB (pSB6A1), ''l4:''PCR of BB (pSB6A1)]] | ||
- | + | ||
PCR shows expected band. | PCR shows expected band. | ||
:=> F1a was cut and gel extracted. | :=> F1a was cut and gel extracted. | ||
<br/> | <br/> | ||
- | + | <div style="clear:both"></div> | |
===Amplification of DelH F1b=== | ===Amplification of DelH F1b=== | ||
====PCR Conditions F1b.W11.D==== | ====PCR Conditions F1b.W11.D==== | ||
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[[File:Heidelberg_20130713 1a 2log 1b 2 BB2.png|200px|thumb|right|'''Fig.11.7'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | [[File:Heidelberg_20130713 1a 2log 1b 2 BB2.png|200px|thumb|right|'''Fig.11.7'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | ||
- | + | ||
PCR did not yield the specific band. | PCR did not yield the specific band. | ||
:=> Use altered PCR conditions. | :=> Use altered PCR conditions. | ||
<br/> | <br/> | ||
- | + | <div style="clear:both"></div> | |
===Amplification of DelH F2=== | ===Amplification of DelH F2=== | ||
====PCR Conditions F2.W11.A==== | ====PCR Conditions F2.W11.A==== | ||
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[[File:Heidelberg_20130713 1a 2log 1b 2 BB.png|200px|thumb|right|'''Fig.11.7'''Gel of PCR of DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | [[File:Heidelberg_20130713 1a 2log 1b 2 BB.png|200px|thumb|right|'''Fig.11.7'''Gel of PCR of DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | ||
- | + | ||
PCR does not show the specific band. | PCR does not show the specific band. | ||
:=> Further optimization. | :=> Further optimization. | ||
<br/> | <br/> | ||
- | + | <div style="clear:both"></div> | |
===Amplification of Backbone=== | ===Amplification of Backbone=== | ||
====PCR Conditions BB.W11.A==== | ====PCR Conditions BB.W11.A==== | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130713 1a 2log 1b 2 BB2.png|200px|thumb|right|'''Fig.11.7'''Gel of PCR of DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | [[File:Heidelberg_20130713 1a 2log 1b 2 BB2.png|200px|thumb|right|'''Fig.11.7'''Gel of PCR of DelH fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' F1b, ''l4:''F2, ''l5:'' BB]] | ||
- | + | ||
PCR does not show the specific band. | PCR does not show the specific band. | ||
:=> Repeat using fresh backbone DNA from miniprep. | :=> Repeat using fresh backbone DNA from miniprep. | ||
<br/> | <br/> | ||
- | + | <div style="clear:both"></div> | |
====Miniprep==== | ====Miniprep==== | ||
* Inocculation of 10 ml LB Amp with DH10ß (pSB6A1-AraC-lacZ) | * Inocculation of 10 ml LB Amp with DH10ß (pSB6A1-AraC-lacZ) | ||
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[[File:Heidelberg_20130713 1a 2log miniprepBB PCRBB.png|200px|thumb|right|'''Fig.11.8'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' Miniprep A of BB, ''l4:''PCR of BB]] | [[File:Heidelberg_20130713 1a 2log miniprepBB PCRBB.png|200px|thumb|right|'''Fig.11.8'''gel of PCR of DelH-fragments (loaded 20 µL) <br> ''l1:''F1a,''l2:''2log, ''l3:'' Miniprep A of BB, ''l4:''PCR of BB]] | ||
- | + | ||
Miniprep shows weak band at ~7.4 Kb. PCR does not show expected band. | Miniprep shows weak band at ~7.4 Kb. PCR does not show expected band. | ||
:=> Prepare new mediprep of backbone (pSB6A1-AraC-lacZ) | :=> Prepare new mediprep of backbone (pSB6A1-AraC-lacZ) | ||
<br/> | <br/> | ||
- | + | <div style="clear:both"></div> | |
===Gibson Assembly Strategy=== | ===Gibson Assembly Strategy=== | ||
[[File:Heidelberg_FinalPlasmid.png|300px|left|thumb|Vector map of the [[:File:Heidelberg_DelH-BB(Gibson-pSB6A1-AraC-lacZ).gb|pHM02:DelH-pSB6A1-AraC-lacZ Gibson-plasmid]].]] | [[File:Heidelberg_FinalPlasmid.png|300px|left|thumb|Vector map of the [[:File:Heidelberg_DelH-BB(Gibson-pSB6A1-AraC-lacZ).gb|pHM02:DelH-pSB6A1-AraC-lacZ Gibson-plasmid]].]] | ||
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! Identifier !! Order date !! Note !! Sequence | ! Identifier !! Order date !! Note !! Sequence | ||
|- | |- | ||
- | | DN01:DelH_f1_PacI_fw || 03-05-2013 || Amplification of DelH F1, with RBS and <br/> adding PacI restriction site || | + | | DN01:DelH_f1_PacI_fw || 03-05-2013 || Amplification of DelH F1, with RBS and <br/> adding PacI restriction site || TTTT TTAATTAA TCACACAGGAAAGTACTAG ATGGACCGTGGCCGCCTGC GCCAAATCG |
|- | |- | ||
- | | DN02:DelH_f1_SalI_rev || 03-05-2013 || Amplification of DelH F1 until SalI <br/>restriction site || | + | | DN02:DelH_f1_SalI_rev || 03-05-2013 || Amplification of DelH F1 until SalI <br/>restriction site || TTTT GTCGACCAACACCTGTGCCTGC |
|- | |- | ||
- | | DN03:DelH_f2_SalI_fw || 03-05-2013 || Amplification of DelH F2 starting at <br/>SalI restriction site|| | + | | DN03:DelH_f2_SalI_fw || 03-05-2013 || Amplification of DelH F2 starting at <br/>SalI restriction site|| TTTT GTCGACTGGATGGAGCCTGGTGAAAG |
|- | |- | ||
- | | DN04:DelH_f2_KpnI_rev || 03-05-2013 || Amplification of DelH F2, adding <br/>KpnII restriction site || | + | | DN04:DelH_f2_KpnI_rev || 03-05-2013 || Amplification of DelH F2, adding <br/>KpnII restriction site || TTTT GGTACC TCAGTCCAGCGCGTACTCCAG |
|- | |- | ||
- | | DN05:AraCbb_KpnI_fw || 03-05-2013 || Amplification of backbone for DelH <br/>(pSB6A1-AraC-lacZ), adding KpnI site || | + | | DN05:AraCbb_KpnI_fw || 03-05-2013 || Amplification of backbone for DelH <br/>(pSB6A1-AraC-lacZ), adding KpnI site || TTTT GGTACC AAAAGAGGAGAAATACTAGATGACCATG |
|- | |- | ||
- | | DN08:AraCbb_PacI_rev || 03-05-2013 || Amplification of backbone for DelH <br/>(pSB6A1-AraC-lacZ), adding PacI site || | + | | DN08:AraCbb_PacI_rev || 03-05-2013 || Amplification of backbone for DelH <br/>(pSB6A1-AraC-lacZ), adding PacI site || TTTT TTAATTAA GCTAGCCCAAAAAAACGGGTATG |
|} | |} | ||
<br/> | <br/> |
Latest revision as of 13:52, 24 October 2013
Contents |
08-07 - 14-07-13
Amplification of DelH F1b
PCR Conditions F1b.W11.A
Reagent | DelH F1b - from purified F1b 05-07 (genome amplified) | DelH F1b - from purified F1b 05-07 (genome amplified) |
---|---|---|
Template | 1 µl of PCR 1b (19 ng/µl) | 1 µl D. acidovorans |
Primer fw 10 µM | 1 µl DelH_EcoRI_fw | 1 µl DelH_EcoRI_fw |
Primer rev 10 µM | 1 µl DelH_f1_SalI_rev 10 | 1 µl DelH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl | 10 µl |
ddH2O | 7 µl | 6 µl |
DMSO | - | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
68 | 5 | |
72 | 2:15 min | |
1 | 72 | 7 min |
1 | 4 | inf |
Result
Expected band: 5 Kb
There is a specific band, but not at 5 Kb.
- => We cannot amplify F1b from a PCR fragment. Use fresh bacteria instead.
Preparation of fresh ACM media
- Mix ingrediants according to Acidovorax complex medium
- Autoclave
- pH adjust to 7,3
- Mix 500 ml media and 6 g agar
- Autoclave again
- Pour plates
Preparation of fresh D. acidovorans
- Inocculate 5 ml ACM media with D. acidovorans
- Incubate ON
- Prepare glycerol stock
- Streak some of culture on plates
PCR Conditions F1b.W11.B
Reagent | DelH F1b | DelH F1b |
---|---|---|
Template | Fresh colony of plate by DN from 10-07 | Fresh colony of plate by DN from 10-07 |
Primer fw 10 µM | 1 µl delH_EcoRI_fw | 1 µl delH_EcoRI_fw |
Primer rev 10 µM | 1 µl delH_f1_SalI_rev | 1 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl | 10 µl |
ddH2O | 7 µl | 6 µl |
DMSO | - | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0.5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5.4 Kb
There is a weak band at the probe 1b WITH DMSO above 5 Kb.
- => Start a new PCR with 50 µl with the same conditions.
PCR Conditions F1b.W11.C
Reagent | DelH 1b |
---|---|
Template | Fresh colony of plate by DN from 10-07 |
Primer fw 10 µM | 2.5 µl delH_EcoRI_fw |
Primer rev 10 µM | 2.5 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 25 µl |
ddH2O | 17.5 µl |
DMSO | 2.5 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0.5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5 Kb.
The expected band at 5 Bk is weak, but visible. Note that there is sample left in the pocket.
- => The fragment was cut and gel extracted.
Generation of DelH plasmid
Restriction Digest
Of fragments F1a, F1b, F2 and backbone.
Fragment | DNA [µl] | ddH2O [µl] | Enzymes [µl] | BSA [µl] | Buffer 4 [µl] |
---|---|---|---|---|---|
F1a (1) | 30 | 7 | 1.5 Pac & EcoRI-HF each | 5 | 5 |
F1a (2) | 30 | 7 | 1.5 Pac & EcoRI-HF each | 5 | 5 |
F1b (11.7 - 20 µl) | 20 | 17 | 1.5 SalI-HF & EcoRI-HF each | 5 | 5 |
F1b (11.7 - 50 µl) | 20 | 17 | 1.5 SalI-HF & EcoRI-HF each | 5 | 5 |
F2 | 20 | 17 | 1.5 SalI-HF & KpnI-HF each | 5 | 5 |
BB (6-7 of IK - 14 ng/µl) | 20 | 17 | 1.5PacI & KpnI-HF each | 5 | 5 |
BB (6-7) | 20 | 17 | 1.5 PacI & KpnI-HF each | 5 | 5 |
Result
Loaded 5 µl of restriction digested fragments on gel. The result stands in the table below:
Fragment | Band | Conclusion | Next step |
---|---|---|---|
F1a purified on 12-07 | strong bands at 1, 2 and 3 Kb weak band at wanted 5 KB | PCR purification of F1a step was not the right purifying step | load complete probe on gel and gel extract |
F1b purified on 12-07 | weak band at 5 Kb | maybe only low concentration | run PCR again |
F2 | no band | maybe too low concentration | run PCR again |
BB | weak band at 7.4 Kb | maybe only low concentration | run PCR again |
Gel Extraction of DelH F1a
Expected band: 5 KB, loaded entire F1a purified on 12-07 on gel
No band visible.
- => Gel extract earlier PCR from 06-07.
Expected band: 5 Kb
PCR shows expected band.
- => F1a was cut and gel extracted.
Amplification of DelH F1b
PCR Conditions F1b.W11.D
Reagent | DelH F1b |
---|---|
Template | 1 µl F1b digested 12-07 |
Primer fw 10 µM | 1 µl delH_EcoRI_fw |
Primer rev 10 µM | 1 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 6µl |
DMSO | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0,5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5.4 Kb
PCR did not yield the specific band.
- => Use altered PCR conditions.
Amplification of DelH F2
PCR Conditions F2.W11.A
Reagent | DelH F2 |
---|---|
Template | 1 µl F2 digested 12-07 |
Primer fw 10 µM | 1 µl delH_f2_SalI_fw |
Primer rev 10 µM | 1 µl delH_f2_KpnI_rev |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 5 s |
30 | 98 | 1 s |
66 | 5 s | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 8 Kb
PCR does not show the specific band.
- => Further optimization.
Amplification of Backbone
PCR Conditions BB.W11.A
Reagent | Backbone |
---|---|
Template | 1 µl backbone digested 12-07 |
Primer fw 10 µM | 1 µl AraCbb_KpnI_fw |
Primer rev 10 µM | 1 µl AraCbb_PacI_rev2 10 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 8 Kb
PCR does not show the specific band.
- => Repeat using fresh backbone DNA from miniprep.
Miniprep
- Inocculation of 10 ml LB Amp with DH10ß (pSB6A1-AraC-lacZ)
- Perform mini prep
PCR Conditions BB.W11.B
Reagent | Backbone |
---|---|
Template | 1 µl Miniprep A of 10-07 |
Primer fw 10 µM | 1 µl AraCbb_KpnI_fw |
Primer rev 10 µM | 1 µl AraCbb_PacI_rev2 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
66 (touchdown -0.5°C) | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results
Expected band: 7.4 Kb, loaded miniprep and PCR product.
Miniprep shows weak band at ~7.4 Kb. PCR does not show expected band.
- => Prepare new mediprep of backbone (pSB6A1-AraC-lacZ)
Gibson Assembly Strategy
Identifier | Order date | Note | Sequence |
---|---|---|---|
DN01:DelH_f1_PacI_fw | 03-05-2013 | Amplification of DelH F1, with RBS and adding PacI restriction site | TTTT TTAATTAA TCACACAGGAAAGTACTAG ATGGACCGTGGCCGCCTGC GCCAAATCG |
DN02:DelH_f1_SalI_rev | 03-05-2013 | Amplification of DelH F1 until SalI restriction site | TTTT GTCGACCAACACCTGTGCCTGC |
DN03:DelH_f2_SalI_fw | 03-05-2013 | Amplification of DelH F2 starting at SalI restriction site | TTTT GTCGACTGGATGGAGCCTGGTGAAAG |
DN04:DelH_f2_KpnI_rev | 03-05-2013 | Amplification of DelH F2, adding KpnII restriction site | TTTT GGTACC TCAGTCCAGCGCGTACTCCAG |
DN05:AraCbb_KpnI_fw | 03-05-2013 | Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding KpnI site | TTTT GGTACC AAAAGAGGAGAAATACTAGATGACCATG |
DN08:AraCbb_PacI_rev | 03-05-2013 | Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding PacI site | TTTT TTAATTAA GCTAGCCCAAAAAAACGGGTATG |