Hi. (i) May I know how long do you store for the leftover lysis...

Thank you Fanglian. Another two questions:
i. How do you ensure that the overnight culture of E. coli is sufficient to provide you the yield between 5 to 10 micrograms?
ii. If I plan not to add in my RNAase into the lysis buffer, do I add RNAase after the step of resuspending the DNA in TE buffer right before checking it on an agarose gel? How long do I need for the incubation?

Hi Henry,

Please see my responses to each of your questions below.

(i) May I know how long do you store for the leftover lysis buffer?

Proteinase K has to be added freshly. Thus, lysis buffer containing proteinase K should not be used next day. Otherwise, it can be kept at room temperature at least 6 months.

(ii) Will it be fine if I use MH broth instead of LB broth?

I never tried MH broth. But, I think it should be fine if your bugs grow happily in the media.

(iii) How much TE buffer do you add for step 18?

50 microliter.
(iv) If I were to determine the concentration of DNA eventually using spectrophotometry, how can I get the dilution factor for the amount of TE buffer added in (iii)?

Well, depending on how you would do the dilution, I guess you have to do your own math to get the answer.

--Fanglian