Question: Making primers for cloning and transfection

Question

[Lu Adr]

Hello! I am designing some primers for the amplification of FUT1 (human), LOCUS: NG_007510 So I need the primers to amplify FUT1 from DNA that will extract from human tissue. My intention is to then add this to a plasmid and transfect this into mammal cells for (hopefully) the expression of the protein in the cell surface. Do I need to design the primers for the whole gene (7348 bp) or just for the CDS (1098 bp) ? The first exon starts at the very start of the gene and ends at the very end so it would be the full 7348 bp. But some papers I've seen (on other proteins) only use the CDS so then they would be missing the 5' and 3' UTRs.

Thank you to anyone that replies!

[Mensur Dlakic]

If you already have a plasmid, especially if under a control of a strong promoter (say SV40 or such), you do not need primers at all. Simply transform the plasmid and the cells will transcribe the gene.

Generally speaking, one clones cDNA sequences rather than genomic DNA sequences. In that case nothing needs to be spliced out.

This is really stretching it as a bioinformatics question.

[Lu Adr]

Well I need the primers to amplify FUT1 after DNA extraction from human cells and then do restriction-insertion cloning to add FUT1 on the plasmid. So I am making primers that will also have restriction enzyme sites. I just want to know whether the whole gene or just CDS should be amplified, so I know what primers to make. I'm sorry if this wasn't the right place to ask this question. I just saw some old similar questions and decided to give it a try

Answer 1

[ChanderKant (CK) Chaudhary]

Hi @Lu Adr

In my opinion, you should make primers from cDNA as it will encompass the UTR region which crucial for gene expression under in vivo conditions. Moreover, I would not recommend gDNA. Though, I have experince only with plant system.

Thank you.

Best,

CK

Answer 2

[jared.andrews07]

Generally, you'd just design them for the open reading frame of interest and clone from cDNA.

But you can get plasmids with the ORF of just about any gene (often with your choice of isoform) commercially at this point. They aren't very expensive (can often be found for $100-500) and often have a variety of backbones you can choose from. You might consider that unless you're really set on this practice.

[Lu Adr]

Thank you! I understand but my supervisor just wants me to do it this way and he's not giving me much direction at all. I just need the primers to amplify FUT1 after DNA extraction from human cells and then do restriction-insertion cloning to add FUT1 on the plasmid.

[jared.andrews07]

Well, definitely CDS only then. And again, you won't want to be amplifying from gDNA. You'll need to isolate RNA and perform reverse transcription to yield cDNA and then use that for your PCR.