DNA binding buffer in Gel extraction kit

[GenePro]

Does anyone know if using more binding buffer than the recommended amount for a DNA gel slice affects the ability of DNA to bind to the silica column in a gel extraction procedure from Qiagen or Fermentas?

[mchlbrmn]

I don't know. My guess is that it would still work. Since you add 3 volumes QG buffer (Qiagen) you have 75% QG final conc. If you add too much QG the maximum will be 100% QG, which is 1.33 x the normal concentration, and that doesn't sound too bad to me since elution is done in low salt. I suppose it would be bad if the agarose could start binding the column, but I have no idea if that's an issue (and doubt it).

[relaxin]

It would not hurt, even with 100% QG buffer. I am sure, because there is an optional step to rinse the spin column with 500 ul of QG buffer after spinning the sample through.

[rahim khan]

Hi,
i am new one in molecular field.i am doing transformation of a gene from bacteria to an expression vector.i did PCR with primers having RE site.then i double digestion.i transfer this PCR product to pBluescript (d.d) and i get positive colonies.then i digest the pBLuescript with RE and did the gel elution using different companies kit.but here i alwys get low concentration (30ng).so any body can help me in this regard?

[relaxin]

I am not sure if you have problem with miniprep DNA extraction or gel extraction of the insert band. How much DNA did you use for the RE digestion? Did you see nice big band of insert on the gel? QIAquick Gel Extraction kit is quite reliable. I have not tried other kits, so I do not know their performance.

[mchlbrmn]

I think we can't tell without details. First, sometimes people don't do the arithmetic and actually 30ng/ul x total volume could be a fair percentage (50%) of the original DNA amount. 30ng/ul x 50 ul = 1.5 ug total purified.
If you started cutting 5 ug 7kb plasmid, cut out a 1kb fragment, then that is 714ng total 1kb band. 50% yield: 357ng. Elute in 50 ul: 7ng/ul.
Maybe you mean 30ng total, but it's not uncommon for people to be surprised.
Sometimes it helps to elute with hot elution buffer, 65C. Two elutions, or passing the elutate through the column a second time can help. There are cases where certain specific sequences don't stick well to the column.
It can also be important not to heat the gel to above the requested temperature (50C?) when dissolving the gel. In some cases it can denature the double stranded DNA so that it does not renature in time to stick to the column.

[rahim khan]

I think we can't tell without details. First, sometimes people don't do the arithmetic and actually 30ng/ul x total volume could be a fair percentage (50%) of the original DNA amount. 30ng/ul x 50 ul = 1.5 ug total purified.
If you started cutting 5 ug 7kb plasmid, cut out a 1kb fragment, then that is 714ng total 1kb band. 50% yield: 357ng. Elute in 50 ul: 7ng/ul.
Maybe you mean 30ng total, but it's not uncommon for people to be surprised.
Sometimes it helps to elute with hot elution buffer, 65C. Two elutions, or passing the elutate through the column a second time can help. There are cases where certain specific sequences don't stick well to the column.
It can also be important not to heat the gel to above the requested temperature (50C?) when dissolving the gel. In some cases it can denature the double stranded DNA so that it does not renature in time to stick to the column.

thanks for helping words.these were really helpful hints for me.i understand the exact calcuation for final yield.i use 5-6ug of Total Plasmid for RE.and i always get 30-36ng/ul with a total volume of 40ul.although i was worried about the concentration but i use the same purified DNA for further ligation with pET28+ and fortunately i succeded in getting the transformed vector.thanks

[rahim khan]

I am not sure if you have problem with miniprep DNA extraction or gel extraction of the insert band. How much DNA did you use for the RE digestion? Did you see nice big band of insert on the gel? QIAquick Gel Extraction kit is quite reliable. I have not tried other kits, so I do not know their performance.

thanks relaxin,i use 5-6ug of total plasmid for RE.and after gel elution i get 30ng/ul DNA.

[relaxin]

For subcloning, I do not measure the DNA concentration after gel extraction. I simply ethanol precipitate the DNA fragment in the presence of glycogen (carrier), and then ligate to 100 ng of cut and dephosphorylated vector.