I have performed an experiment to extract plasmid DNA from a bacterial culture of (1.5 ml) and i had obtained the optical density values as, at (260 nm ) 0.023 and at (280 nm) 0.022 but i am unable to get a clear band while running the gel .Can you please suggest where i have to take care while performing the experiment to avoid errors and what might be the reason for not getting a clear band ?
Unless there is a typo in the question, that A260 is extremely low, and the 260/280 is not consistent with a sample containing significant amounts of RNA or DNA. Those measurements would suggest you had only ~1ng/ul of DNA in your final prep. To get a detectable band you'd need to load in excess of 50ng of DNA (100+ is better for EtBr detection). If the sample did contain clean DNA, the 260/280nm ratio should be closer to 1.8 rather than the ~1 you detected. I think your problem is that you either have no DNA or not enough DNA to detect. I'd recommend trying to re-purify the plasmid from a freshly grown culture.
HI,I hope to help you a little bit.as for me ,if you don't get a clear bond,the main reason is that your plasmid DNA has other thing,such as protein and RNA and so on,and the 260/280 should reach 1.8 around。if you index could not reach this,maybe you should get new plasmid DNA to get a high content.
To extract plasmid from bacteria, you should follow a stander protocol with plasmid extracting kits. Which protocol and kits you used? Skilled hand is one of the important thing. Have you used coloring agent such as ethilium bromide to visualized? You can try again later precisely.
First you OD is very low, probably the bacterial lysis is not good add the fresh NaOH in buffer containing SDS and try to do phenol chloroform extraction of the DNA
Ok, what the concentration of agarose in gel ??
because the plasmid is large in size and it need low cons. agarose in gel to make more space to run through it
You check the purity of plasmid by using A260/280.,but not independently.follow the standard protocol plasmid isolation .
all the best
Hi,
Ur 260 value is too low. anyways, i am not sure whether u r getting a smear or anything else, if in case u r getting a smear it may result due to vigrous vortexing after lysis step which may lead to shearing of plasmid DNA. further u need to isolate plasmid from a higher volume culture and do elution in small qualtity of buffer to increase plasmid concentrtaion so that u reach detectable range on the gel. if u cud attach a gel pic, it wud be easy to interpret
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