Alternative Strategies for Pcr and Cloning and Protein Expression of Ova Science Assignment Help

Assignment Task

Task    

There are many problems with this experiment Outline why the PCR did not work and suggest alternative strategies for PCR and cloning and protein expression of OVA. Basically fix this experiment.

Guidelines for creating a Primer for Protein Expression

1. Find gene sequence - for murine IL2 there was a mRNA sequence - find the coding sequence of the gene. Should start with the ATG start codon.

2. Determine which enzymes in the MCS you want to use and remember to clone the sequence downstream of the promoter and upstream of the poly A signal for expression in an eukaryotic cell line.

3. Create a 5’ end primer aka forward primer.

4. For protein expression in a eukaryotic cell line incorporate a Kozac Sequence GCCACCATG is required. If not present in the database sequence 5’ to the cds append to the primer 5’ to the ATG.

5. Appended the restriction enzyme recognition site (NNNNNNN)5’ to the Kozak sequence.

6. Finally add 6 nucleotide (N) 5’ to the recognition site as described by NEB.

Thus the forward primer will look something like this.

7. Calculate the Tm using the Marmur formula using the nucleotide that will anneal to thetemplate in the 1st round of amplification.

8. Create a reverse primer as above but without the Kozak sequence.

9. Modify primers to ensure melting temperatures are within parameters and all other requirement are met if possible

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