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Team:Heidelberg/Templates/Del week15 AG
From 2013.igem.org
Contents |
07-08-2013
Amplification from FS_02 to FS_26; 16.7 kb
File:20130808 FS02-FS26 FS02-FS11 FS02-FS23 2log.png
Amplifcation of DelAG (FS02-FS26), (FS02-FS11), (FS02-FS23); run at 100 V, 0.8 % gel (TAE)(07.08)
File:20130808 FS02-FS26 FS02-FS11 FS02-FS23 2log cut.png
Amplifcation of DelAG; run at 100 V, 0.8 % gel (TAE)(07.08)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 2 |
| FS_26: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 3:50 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification did not work as expected, nethertheless band was cut out for further amplification with a Re-PCR or test restriction digest.
08-08-2013
Amplification from FS_02 to FS_23; 22.8 kb
- Reaction I
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 4 |
| FS_23: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Reaction II
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 4 |
| FS_13short: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 6:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
10-08-2013
Amplification from FS_02 to FS_26; 16.7 kb
File:20130811 2log AF65const AF65td FS2-26 FS02-23 FS02-23 02-11s 1kbruler.png
Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) ; run at 135 V, 0.8 % gel (TAE)(10.08)
File:20130811 2log AF65const AF65td FS2-26 FS02-23 FS02-23 02-11s 1kbruler cut.png
Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
| Reagent | DelAG | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4.5 µl FS_02 | |||||||||
| Primer rev | 4.5 µl FS_26 | |||||||||
| DMSO | 1 µl | |||||||||
| Phusion Ready Mix | 10 µl | |||||||||
Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 5:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work no product was detectable
- PCR will be repeated with different Primers
Amplification from FS_02 to FS_11_short; 22.8 kb and from FS_02 to FS_23; 22.8 kb
File:20130811 2log AF65const AF65td FS2-26 FS02-23 FS02-23 02-11s 1kbruler.png
Amplifcation of DelAF (02-05) and DelAG (02-26), (02-13), (02-11s) ; run at 135 V, 0.8 % gel (TAE)(10.08)
File:20130811 2log AF65const AF65td FS2-26 FS02-23 FS02-23 02-11s 1kbruler cut.png
Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)
- Reaction
| Reagent | DelAG | |
|---|---|---|
| Template | D.acidovorans SPH-1 colony | |
| Primer fw | 4.5 µl FS_02 | 4.5 µl FS_02 |
| Primer rev | 4.5 µl FS_11_short | 4.5 µl FS_23 |
| DMSO | 1 µl | |
| Phusion Ready Mix | 10 µl | |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work no product was detectable, therefore a Re-PCR using the amplification of DelAG from 10-08-13 will be conducted
11-08-2013
Re-PCR from FS_02 to FS_11; 22.8 kb; 10-08-2013
File:20130812 2log 5xAF(FS02-FS05)@62°Cconst RePCRDelAG(FS02-FS23)withFS02-11sastemplate 1,10Template 1,20Template 1kbruler.png
Amplification of DelAF @62°C const., Re-PCR of DelAG with 1:10 and 1:20 deluted Template from 10.08; run at 135 V, 0.8 % gel (TAE)(10.08)
File:20130812 2log 5xAF(FS02-FS05)@62°Cconst RePCRDelAG(FS02-FS23)withFS02-11sastemplate 1,10Template 1,20Template 1kbruler cut.png
Amplification of DelAF @62°C const., Re-PCR of DelAG with 1:10 and 1:20 deluted Template from 10.08 after cutting; run at 135 V, 0.8 % gel (TAE)(10.08)
- Reaction
| Reagent | DelAG |
|---|---|
| Template | PCR-Product FS02-FS11s from 10-08-2013 |
| Primer fw | 4.5 µl FS_02 |
| Primer rev | 4.5 µl FS_11 |
| Phusion Ready Mix | 10 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work, therefore the template used for the Re-PCR presumably was not the desired amplicon
@62%C2%B0Cconst_RePCRDelAG(FS02-FS23)withFS02-11sastemplate_1,10Template_1,20Template_1kbruler.png){kind=link}
@62%C2%B0Cconst_RePCRDelAG(FS02-FS23)withFS02-11sastemplate_1,10Template_1,20Template_1kbruler_cut.png){kind=link}