Team:HokkaidoU Japan/Notebook/August24
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- | {{Template:HokkaidoU_Japan}} | + | {{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August23|August 23]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August25|August 25]]</div></div> |
+ | |||
+ | =Check to see if digestion visualization Primers Work= | ||
+ | |||
+ | [[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 | ||
+ | |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= | ||
+ | |||
+ | [[Image:HokkaidoU Japan 20100824b.jpg|200px|right|thumb|Electrophoresis to check if various DNA manipulations were successful]] | ||
+ | |||
+ | {| class="protocol" | ||
+ | |'''Lane''' | ||
+ | |'''DNA''' | ||
+ | |- | ||
+ | |2 | ||
+ | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d 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 | ||
+ | |[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d 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 |
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