Team:HokkaidoU Japan/Notebook/August24
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- | = | + | =Check to see if digestion visualization Primers Work= |
- | [[Image:HokkaidoU Japan 20100824a.jpg|200px|right|thumb| | + | |
- | * | + | [[Image:HokkaidoU Japan 20100824a.jpg|200px|right|thumb|Electrophoresis after restriction enzyme digestion]] |
- | {| | + | |
+ | * All 4 of PCR products were purified via Microcon YM-10 | ||
+ | |||
+ | {| class="protocol" | ||
+ | |'''Lane''' | ||
+ | |'''DNA''' | ||
|- | |- | ||
|1 | |1 | ||
Line 19: | Line 24: | ||
|- | |- | ||
|6 | |6 | ||
- | | | + | |Promoter |
|} | |} | ||
+ | Promoter band in lane 6 wasn't visible | ||
+ | * This vector didn't have a primer annealing site for our new primers | ||
- | + | =Check of Ligation Mixtures= | |
- | * | + | TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M] |
+ | * After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added | ||
+ | * Transformed | ||
+ | * incubated in 200 uL of LB medium | ||
+ | * 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol | ||
+ | =Various Checks= | ||
- | + | [[Image:HokkaidoU Japan 20100824b.jpg|200px|right|thumb|Electrophoresis to check if various DNA manipulations were successful]] | |
- | + | ||
- | + | ||
- | + | ||
- | = | + | {| class="protocol" |
- | + | |'''Lane''' | |
- | + | |'''DNA''' | |
- | + | ||
|- | |- | ||
|2 | |2 | ||
- | |λ/''Hin''d III | + | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III] |
|- | |- | ||
|3 | |3 | ||
- | | | + | |Ligation between pSB1C3 vectors |
|- | |- | ||
|4 | |4 | ||
- | | | + | |Product obtained by using digestion visualization primer for 1-2N |
|- | |- | ||
|5 | |5 | ||
- | | | + | |Flow through of digestion visualization primer for 1-2N |
|- | |- | ||
|6 | |6 | ||
- | |λ/''Hin''d III | + | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III] |
|- | |- | ||
|7 | |7 | ||
- | | | + | |Gel extracted pSB1C3 |
|} | |} | ||
- | + | Band weren't visible in lane 3 | |
- | * | + | * Dimers, trimers and etc were hardly visible maybe severely diluted |
- | + | ||
- | * | + | In lane 7 had detectable by very weak band |
+ | * There weren't any problems with gel extraction |
Latest revision as of 07:44, 27 October 2010
Check to see if digestion visualization Primers Work
- All 4 of PCR products were purified via Microcon YM-10
Lane | DNA |
1 | TSUDA Marker I |
3 | RBS |
4 | GFP |
5 | double terminator |
6 | Promoter |
Promoter band in lane 6 wasn't visible
- This vector didn't have a primer annealing site for our new primers
Check of Ligation Mixtures
TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M]
- After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added
- Transformed
- incubated in 200 uL of LB medium
- 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol
Various Checks
Lane | DNA |
2 | λ/Hind III |
3 | Ligation between pSB1C3 vectors |
4 | Product obtained by using digestion visualization primer for 1-2N |
5 | Flow through of digestion visualization primer for 1-2N |
6 | λ/Hind III |
7 | Gel extracted pSB1C3 |
Band weren't visible in lane 3
- Dimers, trimers and etc were hardly visible maybe severely diluted
In lane 7 had detectable by very weak band
- There weren't any problems with gel extraction