Hi, I just have been in fusion cloning.
I want to insert my GOI in pMIG II vector.
So, I amplified my GOI using PCR with 15bp overhang (PFU used), and linearized the pMIG II vector with EcoRI and XhoI.
(PCR fragment and linearized vector were cleaned up using gel elusion kit)
I used Stbl3 for transformation.
And I used in fusion molar ratio calculator to determine the DNA concentration of fragment (1728bp) and vector (6512bp).
But, I failed to get plasmid with my GOI. and I sequenced this failed plasmid and found that this plasmid matched the sequence of the intact pMIG II.
(E.coli did not grow in linearized vector only control)
How do I solve this problem?
Few things to recheck before to revise experiment.
1. check the overhangs of GOI and Plasmid (after restriction with EoRI and Xhol) if the overhangs still exists in the final linearized plasmid.
2. check the antibiotic marker and also competent cell efficiency of plasmid uptake(wild type plasmid)
3. Before transformation, you need to confirm the insertion of GOI into the plasmid through gel electrophoresis and extract the correct size from gel and then try transformation.
4. before transformation you can also sequence your plasmid with insert to find if the insertion is successful or not.
Sayed Abdullah
About answer 3, Do i need to check using in fusion cloning product?
you can run gel with the wt plasmid and Restriction enzyme treated plasmid or linearized plasmid, the wt circular plasmid stay above the linearized one. so you can differentiate between ligated and unlighted plasmid.
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