This thesis report revolved around the cloning process of plasmids. Attempts of cloning the red fluorescent protein mCherry, and the green fluorescent protein EGFP from various plasmids, into other plasmids containing different cell-junction/cytoskeleton plasmids were made. These plasmids were first amplified using PCR, and then cloned using Gibson-Assembly, and then transfected into live HEK293T or MDCK-II cells. After the transfection, the cells were examined in a microscope. The results showed no signal or localization for the cloned plasmids in their respective corresponding channel, 561 nm for the red fluorescent protein mCherry or 488 nm for the green fluorescent protein EGFP. The step that went wrong was the PCR step in the cloning process, since the backbone vector was not successfully amplified. The reasons for this was either that the backbone vector was too long, the primers regions were to rich with Guanine and Cytoseine, or the primers being too long. / Den här tesen kretsade kring kloningsprocessen för plasmider. Det gjordes försök att från plasmider klona in det röda fluorescerande proteinet mCherry, samt det gröna fluorescerande proteinet EGFP in i andra plasmider som innehöll olika cell-junction proteiner. Både det fluorescerande fragmenten och plasmid-vektorerna innehållande cell-junction proteinerna amplifierades med PCR. Sedan gjordes Gibson-Assembly som var själva kloningsmetoden. Efter det transfekterades HEK293T, samt MDCK-II celler med lösningen från Gibson-Assembly kloningen. Dessa celler undersöktes sedan i mikroskop. Resultatet visade inga tydliga signaler varken i 561 nm kanalen (mCherry), eller i 488 nm kanalen (EGFP), vilket betyder att kloningen inte fungerade. Steget som gick fel var PCR-steget i själva kloningsprocessen, då plasmid-vektorerna inte amplifierades. Anledningen till detta var antingen att själva plasmid-vektorerna var för långa, primer regionerna hade för mycket Guanin och Cytosin, eller att alla primers själva var för långa.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-308516 |
| Date | January 2022 |
| Creators | Johansson, Samuel |
| Publisher | KTH, Tillämpad fysik |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | TRITA-SCI-GRU ; 2022:009 |
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