"Question"Why Gel Extraction Kit not working? - (Sep/25/2007 )

As I prepared 4 PCR products and ran them into the gel, I used IQ quick Gel Extraction Kit to purifiy the DNA.
Finally I got nothing in the tubes!!?? What did I go wrong?? Any reason can you think of?? Is this situation always happen? Have you experienced?

I got myself a several possible reasons:
-Incorrected procedure, DNA discarded? (Not likely-I should have followed the protocol)
-Incorrected procedure, Wrong temperature (37C)setup in centrifugation?? (because temperature in centrifugation was not mentioned)
-QI quick Gel Extraction Kit, Solutions expired?? ( how to check? is this important?)
-QI quick Gel Extraction Kit, Solutions previously contaminated? (no idea)
-QI quick Gel Extraction Kit, Is this Kit reliable to all DNA? (my target DNA is around 2000kp)
-QI quick Gel Extraction Kit, Modification of my DNA caused failure of extraction (interaction between column & DNA)?

I am going to purify the PCR products, and I am going to use another technique to replace this Kit!
I am going to extract DNA from PCR products by using QIA quick PCR purification Kit.
Have you guys experienced any problem that I will be facing?

Thanks!!! I am New to BioChem!!Please Tell Me Whatever You Know!!

Things to do to
1: Make sure you have added the 100% ethanol into the wash solution as instructed. The ethanol does not come with the kit. You have to add it yourself. If no ethanol is added to the wash solutioni, the DNA will come off rather then remain bound.

2: Make sure you have add enough QG buffer to the gel (as directed by the handboo). Too much is okay. Too little and DNA will not bind to the column. Remember the gel slab itself has volume.

3: Add 300ul Isopropanol for every 900ul of QG buffer(with gel disolve in it) This helps improve binding to the column.

4: Yeilds can be improved by passing the QG buffer (the orange coloured solution which has your gel slab disolved in) 3 time through the column. This helps gives the column a chance to bind with as much DNA as possible.

5: Heat the elution solution to 65 celsius before using it. If the elution solution is hot, you get more DNA in the eluted off the column. Rather then using a single 50ul, I elute my column first with 20ul, then with a second 30ul. You get just a bit more DNA that way.

Another thing- how intense was your band?? ie how much DNA in the original band?? and what volume did you elute in??
If you have a faint band, and then following gel purification you elute in too big a volume- when you check on a gel, there is so little DNA that it can't be visualised by transillumination.

well i hope you were meaning 2 kpb. Otherwise, column kits are not intented for 2000kpb.
Your solutions can not very alter themselves. But if they are over a year old that should be taken in count.

centrifugation should be done at RT as i know. But maybe your kit need 37° during centrif.
Be sure you didn't overloaded your column. Too much agarose alters both binding and leluting cpacities and you may just loose all pcr product.

You can preequilibrate your column vy the binding buffer without gel.

The intensities of my bands were good enough!!!and I eluted into 50ul. I was sure DNAs were large enough (easily visuallized under UV) but just could not get them from the gel extraction.! So Sad!!

My target genes are 2kbp.
Does the temperature of centrifugation affect much in final product?
If I used 2% of agarose instead of 1% agarose, would this become a big issue in the elution???