Hi
the attach file are plasmid extraction done with alkaline lysis (G- bacteria ) without phenol - chloroform
i need help and suggestion.from right to left (loaded 20ul DNA sample + 5ul Orange G as loading buffer )( 2Hrs 80V ) can anyone help me is the the extraction good in quality ? does my bacteria contain more than one plasmid? which are the bands of plasmids? do i have RNA Contamination
any further opinion well really help me.
Thanks in advance
Hi Saad, it would be better to load a ladder with your extracted plasmid, then If you have the length of your plasmid you can verify that these are your dersired plasmids or not. In addition, before loading on Gel, during Cloning procedures you can chech the OD of your plasmids to avoid contamination with bacteria. Moreover, you check by digestion using enzyme if you want. Remember that, usually plasmids have two bands on gel, the one that is sharper.
Hi,
I suggest using at least 1% agarose gel to obtain clearer bands. Then, you should consider that when running an agarose gel with plasmid DNA extracted from a bacterial colony, it is common to observe more than one DNA band. This is because plasmid DNA can exist in different forms, including the circular shape (supercoiled form), the circular open form, and the linear shape.
Sincerely
Tiziana Irdani Thanks for your information
could you please tell me how to know which one is circular shape (supercoiled form) or linear shape
Generally, the supercoiled form migrates faster than the circular and linear form, which will therefore occur in the lower band of the gel. Then, it will follow the circular and linear, gradually higher and higher.
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