Team:Heidelberg/Templates/Del week15 FG
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==08-08-2013== | ==08-08-2013== | ||
- | ===Restriction digest of fragment from FS_21 to FS_26; 5.5 kb; | + | ===Restriction digest of fragment from FS_21 to FS_26; 5.5 kb; 29-07-2013) with ClaI=== |
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! what !! µl | ! what !! µl | ||
|- | |- | ||
- | | FS_21 to FS_26 ( | + | | FS_21 to FS_26 (29-07-2013) || 10 |
|- | |- | ||
| ClaI || 1 | | ClaI || 1 | ||
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|- | |- | ||
|dd H<sub>2</sub>O || 2.5 | |dd H<sub>2</sub>O || 2.5 | ||
+ | |- | ||
+ | | Expected fragment lengths [bp]|| 2743, 1519, 1208 | ||
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! Fragment !! Primer !! Date PCR !! Concentration | ! Fragment !! Primer !! Date PCR !! Concentration | ||
|- | |- | ||
- | | DelFG || FS21-FS26 | | + | | DelFG || FS21-FS26 || 07-08-2013 || 24.3 ng/µl |
|} | |} | ||
Latest revision as of 19:04, 3 October 2013
Contents |
08-08-2013
Restriction digest of fragment from FS_21 to FS_26; 5.5 kb; 29-07-2013) with ClaI
Incubation at Room temperature for about 4h and at 37°C for 1 hours
what | µl |
---|---|
FS_21 to FS_26 (29-07-2013) | 10 |
ClaI | 1 |
Buffer CutSmart | 1.5 |
dd H2O | 2.5 |
Expected fragment lengths [bp] | 2743, 1519, 1208 |
Results:
- restriction digest of DelFG led to the expected fragment sizes, therefore restriction digest, implies amplification of the correct fragment
- PCR product will be further validated by single read sequencing before Gibson Assembly
Concentration measurement DelFG
Fragment | Primer | Date PCR | Concentration |
---|---|---|---|
DelFG | FS21-FS26 | 07-08-2013 | 24.3 ng/µl |
09-08-2013
Amplification from FS_21 to FS_26; 5.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans SPH-1 (colony-pcr) | 1 |
FS_21: (1/10) | 2 |
FS_26: (1/10) | 2 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 4 |
- Conditions I
Biorad T100 | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
60 | 5 | |
72 | 2:15 | |
1 | 72 | 10 min |
1 | 10 | inf |
Results:
- Amplification of DelFG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit