I have extracted DNA from a cell line. I usually get 5 ug DNA each time when I resuspend in 20 ul water. But this time the max. DNA I obtained was 2ug with same cells, same protocol, same time point, same reagents? what could be the reason? my lysis buffer is 1 year old, phenol we have from himedia around 1 year old and P;C and C:I mixture and sodium acetate 4 months old? Does this less DNA is due to the old reagents? how will we identify whether we have not to use some particular reagents further? For phenol it is said when its color changes from yellow to pink then change it, but the color of our phenol was read from beginning? so how can I identify? how long can we use all of these reagents?
In my lab we usually change the reagents and make fresh ones when they are 6 months to 1 year old. Aqueous buffers and solutions are generally good to use if there is no precipitate.
buffers should not be more than 2 months old. even TE that is used for resuspending DNA solution.
May be your lysate buffer is not working properly which leads to the improper break down of all cells. Old regent affect the quantification of dna. even i have faced the same problem in beginning but once i changed my reagents it was working properly. I use to keep my buffers max for 2-3 months. I agree with sir Artur Burzynski's statement and there are several articles present. Phenol use to get pink or orange when it oxidized which leads to the damage of DNA.
Better to make fresh buffers.Phenol is not necessary to obtain good quality DNA
One year old lysis buffer is no good, prepare little amounts of buffer and refresh it every 2-3 month (only one if you use b-mercaptoethanol). Check the pH of buffers
hey,,,if reagents are older than 1 year replace with new ones. it might be might be you have problem with lysis buffer.
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