Hi all,
I streaked out a glycerol stock, let it grow overnight. Then i picked single colonies and inoculated 2 cultures in LB 500mL each with a single colony using sterile toothpick. I grew them 18 hours and prepped plasmids by boiling lysis followed by phenol/chloroform extraction and EtOH precipitation, then I did a CsCl gradient and another EtOH precipitation. The first plasmid did NOT get a 70% EtOH wash and the second plasmid did get a 70% EtOH wash. Both have good 260/280 and 260/230 by nanodrop. However on the gel they are not running at the same size. This is just straight plasmid no digestion I loaded approximately 2 ug of each plasmid.
Any help or pointers is appreciated! I'm worried about the quality of my plasmid.
Thank you,
David Gorrie
I wouldn't be worried too much about the small difference in migration of your samples. One of them was washed with 70 % EtOH, but not the other, which means that they probably contain different salt concentrations. This will in turn affect their conductivity and their migration. If you wish to be reassured, you could perform a restriction digest on both of them and run them side by side
Some pointers :
- Glycerol stock was not prepared from a clonal population (single colony)
- Growing a single colony in big volumes such as 500 ml is not recommended. Usually, people inoculate a small volume (up to 20ml), let it grow for a few hours or overnight, and then transfer it to the bigger volume (usually using a 1:50 or 1:100 dilution).
I wouldn't be worried too much about the small difference in migration of your samples. One of them was washed with 70 % EtOH, but not the other, which means that they probably contain different salt concentrations. This will in turn affect their conductivity and their migration. If you wish to be reassured, you could perform a restriction digest on both of them and run them side by side
I agree with Pierre, usually plasmids exists in mix supercoiled (dominant form) and single stranded circular plasmid form, thus two bands visible, and there is possibility of third band due to linear or broken plasmid. I am surprised why you choose phenol chloroform method over alkaline lysis method/silica column based kit?
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