Its hard to answer this question without knowing what technique you used for gel extraction. Gel extraction kits normally yield good results providing the protocol is followed closely. If your DNA fragment is too small or too large, it may not bind/elute from the column. So, how was it extracted and what size fragment where you after?
Problem-1.
Due to contamination with DNase that might be present in falcon, micro centrifuge tube.
2. Elution problem through membrane filter.
3. PH difference also causes same problem.
4. You did not add ethanol in wash buffer.
ETC...
First of all, if you are using a extraction kit, you should check that your kit is working properly.
I usually use an alternative method based in DNA precipitation ( it is not a comercial kit). If you are interested on trying that contact me and I will provide you more information.
Thank you all for your excellent input. We used the Feldan Gel Extraction Kit for gel extraction. The size of the plasmid which was isolated is 4700 bp and there were around six visible bands from where we did the gel extraction. After running a 1% gel the bands were quiet visible and cut and the gel extraction followed accordingly to the protocol.
It is a good idea to verify the kit as well.
Thanks!
Mithu
It would be great if i can know about the alternative DNA precipitation, that would be truly helpful.
Thank you!
Mithu
The concentration of plasmid also a crucial point as in gel extraction a large percentage of DNA get lost in each round and It also necessary to elute with the minimum volume of buffer in the last step to increase concentration of eluted plasmid/DNA. It may help.
if you follow the protocol as well , generally it should not happen. As an alternative you can elute DNA from column by using half of the prescribed volume of elution buffer at last step. In such cases you have to use the flow through(elute) again and spin. This will increase yield. now to check on gel use 4-5ul of eluted sample on 1% gel.
A 4.7 kb band will be difficult to isolate with normal kits. They normally work best with 0.5 to 3 kb fragments. An old technique that works well for large fragments:
1. put a small hole in the bottom of a 200 uL microfuge tube with a 20 gage needle
2. Place the tube in a 1.5 mL microfuge tube
3. Add your gel piece (TAE gel) containing the DNA band to the 200 uL tube
4. Centrifuge at full speed in a microcentrifuge for 15 min
5. The gel should remain in the top tube and the liquid containing DNA will pass to the lower tube
6. Ethanol ppt the DNA and resuspend in water or TE
Mahendra Verma is completely right. Even if the gel extraction kits are very convenient, they have one limit : DNA yield after all the purification steps...
If you are using columns, you can also try another kind of kit with silica particles ( QIAEX II from Quiagen for example). A very good point is that you can elute in a smaller volume than "columns kits".
Good luck !
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