Reuse of DNA columns - very less yield (Oct/04/2007 )
Hi all,
I am using the method of reusing the DNA spin column published in Biotechniques by Ranga U
I got a Qiagen tip from my friend. the tip can hold the volume of approximately 15ml.
I treated the tip with 1N HCl for 24hours. Then washed the column with 30ml water and then equilibrated with 30ml QBT buffer.
After the P1, P2 and P3 treatment of 30ml culture pellet as described in Qiagen handbook, loaded the supernatant to the column, washed with 30ml QC buffer and the eluted with 5ml QF buffer. The elute was precipitated with isopropanol, washed with 70% ethanol and then suspended in 200ul water. When 5ul of the solution was checked on the gel, i was able to see only 100ng DNA. i.e the total yield from 30ml culture is approx. 4ug which is rather very low.
The buffers were made exactly as mentioned in Qiagen handbook. But P2 buffer i was making it fresh and then using.
Can someone tell me the way i am doing is correct or whatelse is the possibility of low yield.
Any suggestions are very helpful for me
Thanking you in advance
Leelaram
Which column are these?(mini,midi,maxi??) I have used the same protocol (Ranga Udaykumar) and I got yield of 800ug total DNA from starting 300ml culture (maxi) which is more than what Qiagen people claim! In your protocol check if you have missed QC wash after mill-Q wash during reuse. The used columns (Maxi) I store in 1N HCL and when needed, wash thrice with 30ml milli-q, twice with 30ml QC, equilibrate, bind the sample, wash again, elute, precipitate, wash and resuspend.
Hello!
Did you run a control using a new Qiagen tip using the same sample?
Are you using the method for the first time?
Sorry for questions instead answers, but I am very interested in using this method too...
Mel
(no english speaker 
)
Hi Calvin,
I followed the same protocol as you mentioned. But can you tell me what are the volumes of buffers you used for the columns.
Thank you again
Leelaram
I followed the same protocol as you mentioned. But can you tell me what are the volumes of buffers you used for the columns.
Thank you again
Leelaram
I think one of the elution buffer is not properly made. You sould do a prep using a new colum and a reused colum at the same time (using same buffers) to compare if the problem is the colum or not.