Week 2 — Molecular Reagent Preparation Continued

As I successfully put a piece of gene of interest into one of my donor plasmids, I am able to recombine some of the existed donor plasmids into one. These reactions involves taking 3 donor plasmids and put several pieces of sequences of them (inserts) into one backbone plasmid (vector), which is called LR reactions under the Gateway technology. This week, I set up 4 LR reactions, which are listed as follow.

 

Recombination Reaction:
Reaction 1:
t2k382 (CMV p5E)+ 80807 (GFP)+p3E_MF_001 => t2k394 (pDest)
Reaction 2:
t2k382 (CMV p5E)+80809 (Mem-GFP)+p3E_MF_001 => t2k394 (pDest)
Reaction 3:
t2k327 (UAS p5E)+ 80807 (GFP)+p3E_MF_001 => t2k394 (pDest)
Reaction 4:
t2k327 (UAS p5E)+80809 (Mem-GFP)+ p3E_MF_001 => t2k394 (pDest)
t2k 382 and t2k 327 are 5′ entry plasmids, which contain different promoter regions. One is CMV promoter and one is UAS promoter.┬áThe upstream activation sequence (UAS) is an enhancer that is specific to the GAL4 protein, a protein derived from yeast, which serves as the transcriptional activator in this system.
The promoters for cytomegalovirus (CMV) is commonly used in mammalian expression vectors to drive gene expression and it also works in tadpoles. In other word, if I micro inject one of the CMV promoter contained plasmids into tadpoles, I would be able to see it being expressed in the next day. On the other hand, UAS promoter contained plasmids needs to be co-expressed with other plasmids in order to function.
IMG_0990
After the LR reactions, I performed a series of cell transformation to amply my DNA and did a round of RE diagnosis using enzyme XhoI. The picture above shows part of the results of diagnosis. I then again send the verified plasmids to sequencing and did a midi prep to ensure that I have enough DNA for my experiments in the future. These plasmids are MF_002 to MF_005.

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