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BS934 - Cloning and Expression of Human - Medical Science Assignment Help
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Assignment Task
Your tasks are as follow:
1. Please, design primers for Sanger sequencing to verify the correct sequence (sequencing in both directions) of the CHRAC1-EGFP fusion cDNA in your expression vector. Remember, that a stretch of quality Sanger sequence is only ~ 300 bp and that good sequence only appears ~ 50 bp downstream of the primer. List the primers so they can be ordered from a company. You do not need to verify the sequence of the expression vector backbone itself, only CHRAC1-EGFP and the sequence where it is integrated in the vector.
2. Please, show that your construct is expressed as a fusion protein without disruption of the EGFP reading frame (e.g., show the nucleotide sequence of the transition from CHRAC1 to EGFP and how this would be translated, e.g. 30 nucleotides up- and downstream of the transition point and the translation into protein).
3. Devise a strategy to transfect your expression construct into human cells. Outline the protocol that you would use and justify the choice of the method and protocol.
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