Team:Heidelberg/Templates/DelH week4
From 2013.igem.org
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[[File:Heidelberg_20130523 2log F1a F1a+DMSO F1b 2log pSB1C3 pSB1C3-AraC-lacZ.png|200px|thumb|right|'''Fig.4.1''' PCR ofDelH-fragments and pSB1C3(loaded 20 µL) <br> ''l1:''2log ladder, ''l2-3:'' DelH-F1a without DMSO, ''l4-5:'' DelH-F1a with DMSO, ''l6-7:'' DelH-F1b, ''l8:''2log ladder, ''l9:''pSB1C3 ''l10:'' pSB1C3-AraC-lacZ]] | [[File:Heidelberg_20130523 2log F1a F1a+DMSO F1b 2log pSB1C3 pSB1C3-AraC-lacZ.png|200px|thumb|right|'''Fig.4.1''' PCR ofDelH-fragments and pSB1C3(loaded 20 µL) <br> ''l1:''2log ladder, ''l2-3:'' DelH-F1a without DMSO, ''l4-5:'' DelH-F1a with DMSO, ''l6-7:'' DelH-F1b, ''l8:''2log ladder, ''l9:''pSB1C3 ''l10:'' pSB1C3-AraC-lacZ]] | ||
- | + | ||
The gel shows a lot unspecific bands for both DelH F1a samples. | The gel shows a lot unspecific bands for both DelH F1a samples. | ||
:=> Alter PCR conditions. | :=> Alter PCR conditions. | ||
- | < | + | <div style="clear:both"></div> |
- | + | ||
=== DelH F1b=== | === DelH F1b=== | ||
* The new primer arrived | * The new primer arrived | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130523 2log F1a F1a+DMSO F1b 2log pSB1C3 pSB1C3-AraC-lacZ.png|200px|thumb|right|'''Fig.4.1''' PCR ofDelH-fragments and pSB1C3(loaded 20 µL) <br> ''l1:''2log ladder, ''l2-3:'' DelH-F1a without DMSO, ''l4-5:'' DelH-F1a with DMSO, ''l6-7:'' DelH-F1b, ''l8:''2log ladder, ''l9:''pSB1C3 ''l10:'' pSB1C3-AraC-lacZ]] | [[File:Heidelberg_20130523 2log F1a F1a+DMSO F1b 2log pSB1C3 pSB1C3-AraC-lacZ.png|200px|thumb|right|'''Fig.4.1''' PCR ofDelH-fragments and pSB1C3(loaded 20 µL) <br> ''l1:''2log ladder, ''l2-3:'' DelH-F1a without DMSO, ''l4-5:'' DelH-F1a with DMSO, ''l6-7:'' DelH-F1b, ''l8:''2log ladder, ''l9:''pSB1C3 ''l10:'' pSB1C3-AraC-lacZ]] | ||
- | + | ||
The gel shows a lot unspecific bands for DelH F1b, but also the band at 5 Kb. | The gel shows a lot unspecific bands for DelH F1b, but also the band at 5 Kb. | ||
:=> Band was cut and gel isolated. | :=> Band was cut and gel isolated. | ||
- | < | + | <div style="clear:both"></div> |
===Generation of Backbone pSB6A1-AraC-lacZ=== | ===Generation of Backbone pSB6A1-AraC-lacZ=== | ||
==== Miniprep==== | ==== Miniprep==== | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130521 2log 2xpSB6A1-AraC-lacZ 2log 2xpSB6A1-AraC-lacZ.png|200px|thumb|right|'''Fig.4.2''' PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL) <br> ''l1:''2log ladder, ''l2-3:'' pSB6A1-AraC-lacZ (8),''l4:''2log ladder, ''l5-6:'' pSB6A1-AraC-lacZ (10) <br/> no band visible]] | [[File:Heidelberg_20130521 2log 2xpSB6A1-AraC-lacZ 2log 2xpSB6A1-AraC-lacZ.png|200px|thumb|right|'''Fig.4.2''' PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL) <br> ''l1:''2log ladder, ''l2-3:'' pSB6A1-AraC-lacZ (8),''l4:''2log ladder, ''l5-6:'' pSB6A1-AraC-lacZ (10) <br/> no band visible]] | ||
- | + | ||
The gel shows no bands. | The gel shows no bands. | ||
:=> Perform test restriction to test identity of plasmid. | :=> Perform test restriction to test identity of plasmid. | ||
- | < | + | <div style="clear:both"></div> |
====Restriction Digest A==== | ====Restriction Digest A==== | ||
* Digest with PstI & XbaI of samples 8, 9 and 10 for checking identity of the construct | * Digest with PstI & XbaI of samples 8, 9 and 10 for checking identity of the construct | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130521 2log pSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.3''' PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB6A1-AraC-lacZ (8), ''l3:'' pSB6A1-AraC-lacZ (9), ''l4:'' pSB6A1-AraC-lacZ (10),''l5:''2log ladder, ''l6:'' pSB6A1-AraC-lacZ (8) digested with XbaI & PstI, ''l7:'' pSB6A1-AraC-lacZ (9) digested with XbaI & PstI, ''l8:'' pSB6A1-AraC-lacZ (10)digested with XbaI & PstI, ''l9:''2log ladder, ''l10:'' pSB6A1-AraC-lacZ (8) 1:10 diluted, ''l11:'' pSB6A1-AraC-lacZ (10) 1:10 diluted, ''l12:'' pSB1C3]] | [[File:Heidelberg_20130521 2log pSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.3''' PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB6A1-AraC-lacZ (8), ''l3:'' pSB6A1-AraC-lacZ (9), ''l4:'' pSB6A1-AraC-lacZ (10),''l5:''2log ladder, ''l6:'' pSB6A1-AraC-lacZ (8) digested with XbaI & PstI, ''l7:'' pSB6A1-AraC-lacZ (9) digested with XbaI & PstI, ''l8:'' pSB6A1-AraC-lacZ (10)digested with XbaI & PstI, ''l9:''2log ladder, ''l10:'' pSB6A1-AraC-lacZ (8) 1:10 diluted, ''l11:'' pSB6A1-AraC-lacZ (10) 1:10 diluted, ''l12:'' pSB1C3]] | ||
- | + | ||
The gel shows a band at 5 Kb. | The gel shows a band at 5 Kb. | ||
:=> Colonies 8, 9 and 10 are not the desired construct. | :=> Colonies 8, 9 and 10 are not the desired construct. | ||
- | < | + | <div style="clear:both"></div> |
====Restriction Digest B==== | ====Restriction Digest B==== | ||
* Digest with KpnI of samples 8, 9, 10 for checking length of the construct and KpnI and BamHI to check for identity. | * Digest with KpnI of samples 8, 9, 10 for checking length of the construct and KpnI and BamHI to check for identity. | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | [[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | ||
- | + | ||
:=> Colonies 8, 9 and 10 are not desired construct. Pick new colony from pSB1C3-AraC(6)-lacZ and test digest. | :=> Colonies 8, 9 and 10 are not desired construct. Pick new colony from pSB1C3-AraC(6)-lacZ and test digest. | ||
- | |||
+ | <div style="clear:both"></div> | ||
====Miniprep==== | ====Miniprep==== | ||
A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed. | A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed. | ||
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<br/> | <br/> | ||
[[File:Heidelberg_20130523 2log pSB1C3-AraC-lacZ-digested.png|200px|thumb|right|'''Fig.4.5''' Test digest of pSB1C3-AraC-lacZ with EcoRI & PstI (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' pSB1C3-AraC-lacZ ]] | [[File:Heidelberg_20130523 2log pSB1C3-AraC-lacZ-digested.png|200px|thumb|right|'''Fig.4.5''' Test digest of pSB1C3-AraC-lacZ with EcoRI & PstI (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' pSB1C3-AraC-lacZ ]] | ||
- | + | ||
The gel showed again these three bands (2.070 and 3.360 and ~4 Kb) | The gel showed again these three bands (2.070 and 3.360 and ~4 Kb) | ||
:=> Colonies 8, 9 and 10 are not the desired construct. | :=> Colonies 8, 9 and 10 are not the desired construct. | ||
- | < | + | <div style="clear:both"></div> |
- | + | ||
===Generation of Backbone pSB1C3-AraC-lacZ=== | ===Generation of Backbone pSB1C3-AraC-lacZ=== | ||
====Test Restriction Digest==== | ====Test Restriction Digest==== | ||
Line 299: | Line 297: | ||
<br/> | <br/> | ||
[[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | [[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | ||
+ | |||
+ | :=> pSB1C3-AraC(6)-lacZ is not desired construct. Pick new colony and test digest. | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
- | |||
- | |||
====Miniprep==== | ====Miniprep==== | ||
A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed. | A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed. | ||
Line 328: | Line 326: | ||
<br/> | <br/> | ||
[[File:Heidelberg_20130524 2log pSB1C3-AraC-lacZ-digested pSB6A1-AraC-lacZ-digested.png|200px|thumb|right|'''Fig.4.6''' BB digested with EcoRI & PstI (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' digested pSB1C3-AraC-lacZ, ''l3:'' digested pSB6A1-AraC-lacZ]] | [[File:Heidelberg_20130524 2log pSB1C3-AraC-lacZ-digested pSB6A1-AraC-lacZ-digested.png|200px|thumb|right|'''Fig.4.6''' BB digested with EcoRI & PstI (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' digested pSB1C3-AraC-lacZ, ''l3:'' digested pSB6A1-AraC-lacZ]] | ||
- | + | ||
Gel showed again the three bands (2070 and 3360 and ~4 Kb). | Gel showed again the three bands (2070 and 3360 and ~4 Kb). | ||
:=> pSB1C3-AraC(6)-lacZ is not desired construct. Start assembly all over again. | :=> pSB1C3-AraC(6)-lacZ is not desired construct. Start assembly all over again. | ||
- | < | + | <div style="clear:both"></div> |
====Test Restriction Digest pSB6A1-J04450==== | ====Test Restriction Digest pSB6A1-J04450==== | ||
pSB6A1-J04450 was test digested to check for identity. | pSB6A1-J04450 was test digested to check for identity. | ||
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====Result==== | ====Result==== | ||
[[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | [[File:Heidelberg_20130522 2log pSB1C3 pSB6A1 3xpSB6A1-AraC-lacZ 8 9 10.png|200px|thumb|right|'''Fig.4.4''' BB pSB6A1-AraC-lacZ test digested (loaded 50 µL) <br> ''l1:''2log ladder, ''l2:'' pSB13-AraC-lacZ (8), ''l3:'' pSB6A1, ''l4-6:'' pSB6A1-AraC-lacZ digested with BpsI, ''l7:''2log ladder, ''l8:'' pSB13-AraC-lacZ (8), ''l9:'' pSB6A1, ''l10-12:'' pSB6A1-AraC-lacZ digested with BpsI & BamHI]] | ||
- | |||
{| class="wikitable" style="float:left" | {| class="wikitable" style="float:left" | ||
|- | |- | ||
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| pSB6A1-AraC-lacZ (8, 9, 10) || 7.388 Kb (linearized) || 6 Kb & 10 Kb || 4.607 Kb & 2.781 Kb|| 6Kb & 10 Kb | | pSB6A1-AraC-lacZ (8, 9, 10) || 7.388 Kb (linearized) || 6 Kb & 10 Kb || 4.607 Kb & 2.781 Kb|| 6Kb & 10 Kb | ||
|} | |} | ||
- | + | ||
This means: | This means: | ||
* The chloramphenicol backbone has the right length. | * The chloramphenicol backbone has the right length. | ||
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:=> Something is not ok with this ligated construct and we have to start all over again. | :=> Something is not ok with this ligated construct and we have to start all over again. | ||
<br/> | <br/> | ||
+ | <div style="clear:both"></div> |
Revision as of 18:25, 23 October 2013
Contents |
20-05 - 26-05-13
DelH Fragment F1a
- The new primer arrived.
PCR Conditions F1a.W4.A
Reagent | DelH F1a | DelH F1a |
---|---|---|
Expected length [Kb] | 5 | 5 |
Template | 1 µl D. acidovorans | 1 µl D. acidovorans |
Primer fw 10 µM | DN01 | DN01 |
Primer rev 10 µM | DN08 | DN08 |
Phusion Master Mix (2x) | 25 µl | 25 µl |
DMSO | 2.5 µl | - |
ddH2O | 20,5 | 23 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
16 | 98 | 1 s |
62 | 5 s | |
72 | 2:00 min | |
1 | 72 | 2:30 min |
1 | 4 | inf |
Results
Expected band: 5 Kb
The gel shows a lot unspecific bands for both DelH F1a samples.
- => Alter PCR conditions.
DelH F1b
- The new primer arrived
PCR Conditions F1b.W4.A
Reagent | DelH F1b |
---|---|
Expected length [Kb] | 5 |
Template | 1 µl D. acidovorans |
Primer fw 10 µM | DN07 |
Primer rev 10 µM | DN02 |
Phusion Master Mix (2x) | 25 µl |
DMSO | - |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
16 | 98 | 1 s |
62 | 5 s | |
72 | 2:00 min | |
1 | 72 | 2:30 min |
1 | 4 | inf |
Result
Expected band: 5 Kb
The gel shows a lot unspecific bands for DelH F1b, but also the band at 5 Kb.
- => Band was cut and gel isolated.
Generation of Backbone pSB6A1-AraC-lacZ
Miniprep
- From three different clones, 2ml of pSB6A1-AraC-lacZ (6) in LB Amp were minipreped and eluted in 30 µl ddH2O. Additionally, a glycerol stock was prepared.
Result
Sample | Concentration [ng/µl] |
---|---|
8 - pSB6A1-AraC-lacZ (6) | 63 |
9 - pSB6A1-AraC-lacZ (6) | 61 |
10 - pSB6A1-AraC-lacZ (6) | 62 |
PCR Conditions BB.W4.A
Reagent | 8 - pSB6A1-AraC-lacZ (6) | 9 - pSB6A1-AraC-lacZ (6) |
---|---|---|
Expected length [Kb] | 7.382 | 7.382 |
Template | 1 µl of 1:10 diluted 8 | 1 µl of 1:10 diluted 9 |
Primer 10 µM fw | 0.5 µl | |
Primer 10 µM rev | 0.5 µl | |
Phusion Master Mix (2x) | 25 µl | 25 µl |
DMSO | - | - |
ddH2O | 23 µl | 23 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
16 | 98 | 1 s |
66↓ | 5 s | |
72 | 3:00 min | |
14 | 98 | 1 s |
63 | 5 s | |
72 | 3:00 min | |
1 | 72 | 5:00 min |
1 | 4 | inf |
Results
Expected band: 7.4 Kb
The gel shows no bands.
- => Perform test restriction to test identity of plasmid.
Restriction Digest A
- Digest with PstI & XbaI of samples 8, 9 and 10 for checking identity of the construct
Reagent | Amount [µl] |
---|---|
DNA | 10 |
Enzymes | 1.5 each |
Buffer NEB 3 (10x) | 5 |
BSA (10x) | 5 |
ddH2O | 27 |
- Incubated for 1 h at 37°C
Result
Expected bands: 3360 and 4022 bp
The gel shows a band at 5 Kb.
- => Colonies 8, 9 and 10 are not the desired construct.
Restriction Digest B
- Digest with KpnI of samples 8, 9, 10 for checking length of the construct and KpnI and BamHI to check for identity.
Reagent | Amount [µl] |
---|---|
pSB6A1-AraC-lacZ (8, 9, 10) | 5 |
Enzymes | 1.5 KpnI |
Buffer NEB 3 (10x) | 5 |
BSA (10x) | 5 |
ddH2O | 33,5 |
Expected bands | 7.381 bp |
Reagent | Amount [µl] |
---|---|
pSB6A1-AraC-lacZ (8, 9, 10) | 5 |
Enzymes | 1.5 KpnI & 1,5 BamHI |
Buffer NEB 3(10x) | 5 |
BSA (10x) | 5 |
ddH2O | 32 |
Expected bands | 4.607 bp & 2.781 bp |
Result
- Expected bands: 7.381 bp and 4.607 bp & 2.781 bp
- => Colonies 8, 9 and 10 are not desired construct. Pick new colony from pSB1C3-AraC(6)-lacZ and test digest.
Miniprep
A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.
Result
pSB6A1-AraC(6)-lacZ construct : 94 [ng/µl]
Test Restriction Digest
The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.
Component | Amount [µl] |
---|---|
DNA | 5 |
BSA (10x) | 5 |
NEB2 buffer (10x) | 5 |
Enzymes (EcoRI-HF and PstI) | 1.5 each |
ddH2O | 32 |
- Incubated 1 h at 37°C
Result
Expected bands: ? Kb
The gel showed again these three bands (2.070 and 3.360 and ~4 Kb)
- => Colonies 8, 9 and 10 are not the desired construct.
Generation of Backbone pSB1C3-AraC-lacZ
Test Restriction Digest
In order find out why pSB6A1-AraC-lacZ shows wrong restriction pattern, parental pSB1C3-AraC-lacZ was restriction digested using KpnI as well as KpnI & BamHI.
Reagent | Amount [µl] |
---|---|
pSB1C3-AraC-lacZ | 5 |
Enzymes | 1.5 KpnI |
Buffer NEB 3(10x) | 5 |
BSA (10x) | 5 |
ddH2O | 33.5 |
Expected band | 5,436 bp (linerized) |
Reagent | Amount [µl] |
---|---|
pSB1C3-AraC-lacZ | 5 |
Enzymes | 1.5 KpnI & 1.5 BamHI |
Buffer NEB 3(10x) | 5 |
BSA (10x) | 5 |
ddH2O | 32 |
Expected bands | 4.906 bp & 530 bp |
Result
Expected bands: 5.436 bp and 4.906 bp & 530 bp
- => pSB1C3-AraC(6)-lacZ is not desired construct. Pick new colony and test digest.
Miniprep
A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.
Result
pSB1C3-AraC(6)-lacZ construct : 203 [ng/µl]
Test Restriction Digest
The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.
Component | Amount [µl] |
---|---|
DNA | 3 |
BSA (10x) | 5 |
NEB2 buffer (10x) | 5 |
Enzymes (EcoRI-HF and PstI) | 1.5 each |
ddH2O | 34 |
- Incubated 1 h at 37°C
Result
Expected band: ? Kb
Gel showed again the three bands (2070 and 3360 and ~4 Kb).
- => pSB1C3-AraC(6)-lacZ is not desired construct. Start assembly all over again.
Test Restriction Digest pSB6A1-J04450
pSB6A1-J04450 was test digested to check for identity.
Reagent | Amount [µl] |
---|---|
pSB6A1-J04450 | 10 |
Enzymes | 1.5 KpnI |
Buffer NEB 3 (10x) | 5 |
BSA (10x) | 5 |
ddH2O | 28.5 |
Expected band | 5,091 bp - Linearized |
Reagent | Amount [µl] |
---|---|
pSB6A1-J04450 | 10 |
Enzymes | 1.5 KpnI & 1.5 BamHI |
Buffer NEB 3 (10x) | 5 |
BSA (10x) | 5 |
ddH2O | 27 |
Expected bands | 4,906 bp & 530 bp |
Result
Sample | Expected bands with KpnI | Bands on gel with KpnI | Expected bands with KpnI & BamHI | Bands on gel with KpnI & BamHI |
---|---|---|---|---|
pSB1C3-AraC-lacZ | 5.436 Kb (linerized) | 10 Kb | 4.906 Kb & 530 Kb | no band |
pSB6A1-J04450 | 5.091 Kb (linearized) | no band | 5.091 Kb (linearized) | no band |
pSB6A1-AraC-lacZ (8, 9, 10) | 7.388 Kb (linearized) | 6 Kb & 10 Kb | 4.607 Kb & 2.781 Kb | 6Kb & 10 Kb |
This means:
- The chloramphenicol backbone has the right length.
- The digested chloramphenicol backbone is also ok.
- The digested fragment pSB6A1-AraC-lacZ (with PstI & EcoRI-HF) showed the 2 expected bands at 2,070 bp and 3,360 bp, but another band around ~4 Kb.
- => Something is not ok with this ligated construct and we have to start all over again.