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Team:Heidelberg/Templates/Del week13 G
From 2013.igem.org
(Difference between revisions)
(→Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with BglII) |
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==22-07-2013== | ==22-07-2013== | ||
| - | ===Re-PCR from FS_10 to FS_23; 3.3 kb; | + | ===Re-PCR from FS_10 to FS_23; 3.3 kb; 13-07-2013=== |
[[File:Heidelberg_20130723 log2 G8to23long+ G8to23- G10to23besch.png |200px|thumb| Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07); run at 100 V, 0.8 % gel (TAE)]] | [[File:Heidelberg_20130723 log2 G8to23long+ G8to23- G10to23besch.png |200px|thumb| Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07); run at 100 V, 0.8 % gel (TAE)]] | ||
[[File:Heidelberg_20130723 log2 G8to23long+ G8to23- G10to23 cut.png |200px|thumb| Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07) cut; run at 100 V, 0.8 % gel (TAE)]] | [[File:Heidelberg_20130723 log2 G8to23long+ G8to23- G10to23 cut.png |200px|thumb| Re-PCR of DelG (10-23), Amplification of DelG (8-23) (22.07) cut; run at 100 V, 0.8 % gel (TAE)]] | ||
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! what !! µl | ! what !! µl | ||
|- | |- | ||
| - | | Fragment FS_10 to FS_11_short ( | + | | Fragment FS_10 to FS_11_short (13-07-2013) || 1 |
|- | |- | ||
| FS_10: (1/10) || 2 | | FS_10: (1/10) || 2 | ||
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==26-07-2013== | ==26-07-2013== | ||
| - | ===Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; | + | ===Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with BglII=== |
[[File:Heidelberg_20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII OPEcoRIHFbesch.png|150px|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|150px|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)]] | ||
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! what !! µl | ! what !! µl | ||
|- | |- | ||
| - | | FS_08 to FS_23 cut1 ( | + | | FS_08 to FS_23 cut1 (23-07-2013) || 15 |
|- | |- | ||
| BglII || 0.5 | | BglII || 0.5 | ||
Latest revision as of 18:29, 3 October 2013
Contents |
22-07-2013
Re-PCR from FS_10 to FS_23; 3.3 kb; 13-07-2013
- Reaction
| what | µl |
|---|---|
| Fragment FS_10 to FS_11_short (13-07-2013) | 1 |
| FS_10: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
--> First cycles with wrong program
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:10 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:10 min | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
Results:
- Re-Amplification of DelG lead to the expected fragment but also to a smear, therefore fragment will not be used for Gibson Assembly but restriction digests to validate the product.
Amplification from FS_08 to FS_23; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/- |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG worked, leading to the expected product as well one other unexpected band.
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification from FS_08 to FS_11s; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_11_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
23-07-2013
Amplification from FS_08 to FS_23; 6.5 kb
_psb6A1(3)_4xFS08toFS23_empty_log2_1.png){kind=spacer}
_psb6A1(3)_4xFS08toFS23_empty_log2_cut_1.png){kind=spacer}
4x 20µl
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- Gel to proof quality of gel extraction was run
26-07-2013
Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with BglII
Incubation at 37°C for 1hour 45min
| what | µl |
|---|---|
| FS_08 to FS_23 cut1 (23-07-2013) | 15 |
| BglII | 0.5 |
| Buffer 3.1 | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 3291, 1936, 1303 |
Results:
- Restriction digest of DelG (FS_08 to FS_23) was successful though one unexpected band (presumably carry over) appeared
- Construct will be further validated by sequencing before potential Gibson Assembly
