Is it possible to harvest E.coli cells for purification of DNA from LB agar and - (May/15/2006 )
i'm a student from a polytechnic in singapore and i'm currently doing my final year project. tomorrow, i'm supposed to extract and purify genomic DNA from E.coli cells. The supervisor in charge said to grow the cells on LB agar. what got us stumped was how to harvest the cells to bring it in the right form to purify and extract the DNA. the protocol said to harvest cells (maximum 2 x 10^ 9) in a microcentrifuge tube by centrifuging for 10 min. are we to take a loop of cells from the LB agar, and suspend in some solution and then centrifuge? but what kind of solution to use exactly? sterile water or buffer? if it is a buffer, then what sort? would we need to obtain the OD first? please advice us! 
I would have started from a 3 - 5 ml overnight culture in L broth, but if you've grown your cells on a plate, you can achieve approximately the same result by pipetting 3 - 5 ml of L-broth (or PBS) onto the plates, harvest (resuspend) the cells with an alcohol-flamed glass spreader, and pipete the liquid into a test tube. Vortex the tube briefly to evenly resuspend the cells, and withdraw an ml into a microfuge tube. From there, you can proceed as your protocol dictates.
What method are you using to isolate genomic DNA? Do you mean to isolate genomic DNA (chromosome plus resident plasmids, if any), or are you trying to isolate chromosomal DNA?
I wouldn't get too hung up on the cell count -- I've never known a chromosomal DNA isolation method that dictates a maximum number of cells...
Aiya,
Don't listen to your supervisor lah. Grow a loopful in 15-20ml LB broth overnight and just extract using a kit. No problem one
Chris 
hm okay we've decided to transfer the cells to LB broth (around 20ml). thanks for the help!
well we are supposed to isolate gDNA using a QIAGEN kit. also how do we optimise the gDNA? in our protocol sketch provided by our supervisor, it said : Isolate gDNA (QIAGEN kit) – optimisation, RNase A, run gel.
any ideas? thanks!
well we are supposed to isolate gDNA using a QIAGEN kit. also how do we optimise the gDNA? in our protocol sketch provided by our supervisor, it said : Isolate gDNA (QIAGEN kit) – optimisation, RNase A, run gel.
any ideas? thanks!
Dear LLN,
Isolate the gDNA according to the protocol. There is no need to determine cell number or anything. JUz dump all the 20ml into your extraction. I dunno what you mean by optimization. Could you ask your supervisor exactly what he means? Coz extract DNA is simply extract DNA. There is no need to optimize anything unless this is the first time anyone has done E coli extraction at your place. RNase A is to destroy RNA in your DNA that you have extracted. But depending on what is your application with tha DNA the RNAse step is not really necessary. Hope 4 your success.
Chris
helloooo. we have already harvested the E coli cells and are planning to use DNeasy kit to isolate the gDNA and after that run the gel soon. but we are now unsure of the percentage of agarose gel that we are supposed to use. In the Dneasy kit protocol, it said that it extracts up to 50kb of DNA and predominatly 30kb of smaller fragments. Does this mean that we have to prepare 0.4% agarose gel to run our sample or should it be 1%?
thanks alot! 