We would like to use an enzyme tagged with eGFP and eGFP-tagged alpha synuclein at the same time to observe them under confocal microscopy. However, this will give wrong readings, so we would like to replace eGFP with mCherry. There is a research used the same way but without a detailed explanation.
I will add the link of this research below:
Dinter E, Saridaki T, Nippold M, Plum S, Diederichs L, Komnig D, Fensky L, May C, Marcus K, Voigt A, Schulz JB, Falkenburger BH. Rab7 induces clearance of α-synuclein aggregates. J Neurochem. 2016 Sep;138(5):758-74. doi: 10.1111/jnc.13712. Epub 2016 Aug 4. PMID: 27333324.
thanks in advance.
Mohamed Khashan If you have a fragment containing mCherry cDNA, analyze the respective cDNA sequences and check if there are any restriction sites you can use for switching the inserts. If none available, design adapter primers to introduce them by PCR and/or site directed mutagenesis.
Alternatively, consider gene synthesis for your whole construct (which has become quite affordable, when you take in account time and cost of labor). When designing gene sequences, optimize codon usage for your target organism.
If you can work with fixed cells, consider immunostaining instead.
Mohamed Khashan
As has been mentioned, a simple sub-cloning should do the trick. The caveat being that you have some DNA carrying the coding sequence of mCherry.
Cloning options:
1. Restriction enzyme-based cloning
2. Restriction-free cloning - rf-cloning.org (this works great and is often cheaper, especially if you don't want to order restriction enzymes)
3. Gibson cloning
Happy cloning!
I think you need to see for restriction sites in both plasmids and then using restriction sites common in both you can cut out your insert from eGFP and ligate it in mCherry plasmid
Wolfgang Schechinger Marli Vlok Noorul Ain thank you very much I think that would help, I will try and give a feedback for more benefit.
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