Dear all,
How I can clone two genes in pcDNA vector, simultaneously (one of them is target gene with 500 bp length and one of them is GFP). Also, how we can transfect the vector into SH-SY5Y cell line. I appreciate any comment and idea, tricks, important details and any point that can help me with the procedure.
Best regards
hi
your question is very general.
for transfection "jetPEI" reagent is good choice .
https://www.polyplus-transfection.com/products/jetpei/
1- you can express your target gene in pEGFP-N1 vector (EGFP as reporter gene )
http://ibrc.ir/index.aspx?fkeyid=&siteid=1&pageid=311&siteid=1
http://www.tsv-co.com/product2-3.html
2- you can 2 PCR products fused by soeing PCR method and then one PCR product include your "target gene+GFP " clone in pCDNA3.1+
Article Gene Splicing by Overlap Extension: Tailor-Made Genes Using ...
good luck
In addition to the answer above, you can also use the NEB HiFi assembly kit and order your two fragments as double stranded DNA from a vendor such as IDT.
https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Product%20Information
If each of your double stranded DNA fragments contain 15-30 bp of overlap with your restriction enzyme digested vector / other fragment, then you can combine your digested vector and two fragments in one reaction. Using this method bypasses the need for an overlap extension PCR, although you will be spending more money. Ordering dsDNA fragments from oligo vendors can also be problematic if your inserts contain repetitive sequences like viral ITR / LTRs. Additionally, you want your restriction enzymes to be blunt or have 3' overhangs since the HiFi kit is basically a Gibson assembly.
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