I don't have 12000 g centrifuge. I did 3220g 4 times.
It took me some time to add lysate to the column due to debris.
According to the protocol, the total volume after adding Elution Buffer and isopropanol = 8.5ml. But I only had 8ml. I guess some got stuck within the column.
It took me some time to centrifuge after this.
I dont know if these are the reasons for low results.
I am doing this for the 2nd time.
Nanodrop for plasmid DNA
ng/ul A260/A280 A260/A230
A-+ PLASMID 18.8 2.13 0.73
B-+ PLASMID 40.5 2.22 1.77
C-E.coli with just AmpR 17.3 2.71 0.25
Can someone help me as I am going to repeat this again?
Thank you
High-speed centrifugation helps to remove most contaminants (proteins, endotoxins, and phenol) to achieve the ideal A260/A280 ratio. Preheating the elution buffer to 65°C before loading it into the column can help increase the yield. Ensure the bacterial OD is higher than 1.
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