Hi All
I have a technical question regarding the transfection control. For the WT control (where we use the parent plasmid for cloning) Should i make a PCR reaction for it , transformation , miniprep and used for transfection ? or use it directly in transfection? I am just starting to learn this technique and i will appreciate any help.
Thank you
You should varant to have enough plasmid.
If you have enough, you can use it directly without multiplication via PCR.
If you have only a small amount of the plasmid, I would propose tio perform a multiplication of the plasmid befor using it for your experiments.
You can amplify a sequence wt and your sequence with the mutation then clone into a expression vector with a tag (flag or myc, Nter or Cter). To check if you are in good conditions for transfection use a vector that express GFP.
For your plasmid : do a transformation for amplification, check by PCR your colony, make a miniprep
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