I want to get 5-10 micrograms of linearized plasmid for Pchia pastoris' electrophoretic transfer, 20 micrograms of plasmid is linearized and then dephosphorylated, 2.5 volume of ethanol is added to precipitate plasmid, after that double distilled water is added to dissolve the dried precipitation, and finally the weight of plasmid is calculated by Invitrogen's Qubit assay,.However, to my disappointment, I only get 2 micrograms of plasmid is . Please help me to solve this problem.
You might add NaCl to 500mM or NaAcetate prior to adding the the alcohol. Isopropanol also tends to increase yields compared to ethanol in my experience.
Other tricks to improve yield: Incubate longer prior to centrifugation. Chill the sample prior to centrifugation (overnight at -20*C is doable). Centrifuge longer. Centrifuge at higher RPM. Centifuge at a lower temperature.
I've had good luck using glycogen as a carrier to improve precipitation and reduce the change of losing the pellet. Some people use a product called GlycoBlue for the same purpose (https://www.thermofisher.com/order/catalog/product/AM9516).
I'm not sure if that would interfere with Qubit, but it does not interfere with NanoDrop.
Hi
You may have lost the plasmid during purification or it may have not precipitated effectively...do it more carefully or use some carrier molecule like LPA or tRNA or glycogen for effective precipitation...
You might add NaCl to 500mM or NaAcetate prior to adding the the alcohol. Isopropanol also tends to increase yields compared to ethanol in my experience.
Other tricks to improve yield: Incubate longer prior to centrifugation. Chill the sample prior to centrifugation (overnight at -20*C is doable). Centrifuge longer. Centrifuge at higher RPM. Centifuge at a lower temperature.
I've had good luck using glycogen as a carrier to improve precipitation and reduce the change of losing the pellet. Some people use a product called GlycoBlue for the same purpose (https://www.thermofisher.com/order/catalog/product/AM9516).
I'm not sure if that would interfere with Qubit, but it does not interfere with NanoDrop.
Hi Lide,
I always use the following procedure and I usually get yields of plasmid:
1. Ethanol precipitation:
i. Precipitate the DNA by addition of 10% volume of 3 M sodium acetate and 200% volume of 10,000 g for 15 minutes.
vi. Remove the supernatant and allow to air dry in a sterile environment.
vii. Once dry resuspend in 10 µl/transfection water.
Good luck,
LUIS
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