your 260/280 ratios are too high so try purifying less sample and add an extra wash step of the column to get rid of Guanidine thiocyanate denaturant from the column. When eluting the dna use 50ul of hot (70c) elution buffer which will increase the yield.

How much dna do ou need to make a probe.....you have 128ng/ul and 50 ul which seems quite a lot even if some of it is guanidine salts

Hello,

Your 2 ratios 260/280 and 260/230 are greater than 2, which means that your sample is contaminated, respectively, by proteins and sugars, so you have a problem in the purification step.

I don't know how much you need for the rest, but maybe it's this contamination by proteins and sugars that gave you less quantity (inversely proportional).

The concentrations are pretty decent, how much are you wanting? The contamination issue can be helped by an additional wash step. Also, if it's a column, try to avoid transferring over any cell debris after lysis and clearing.

Thank you very much for your kind response. Previously I have been using plasmid with the concentration above 200. This time I will try to make probe with this concentration and if it doesn't works then I will do extraction again.

I agree with Paul Rutland that your 260/280 ratios are too high, what this means is that the 260 measurement is not going to be accurate. You have no idea what your DNA concentration will be from this measurement. You can double check on a gel just to be sure of whether you have plasmid or not.

Tiangen miniprep kit always give high concentration plasmid. Btw I think your plasmid concentration should not be your concern. But the a260/280 ratio should be your real concern cause it is too high, it indicates that your sample quality is so bad.

1) try to resuspend completely by vortex

2)ensure rnase is added and store under 4c

3) after adding lysis buffer, mix gently otherwise your dna will crack

4)(not a must) run a sds page and extract plasmid afterwards

As a note, running your plasmid on a gel with a good DNA ladder can also give you a rough estimate of the yield. Many DNA ladders include the amount of DNA in each band of a standard loading along with the DNA band sizes. For example, NEB has a 1 KB DNA ladder, you load 5 ul prepared ladder, then find the band size closest to your plasmid. In this example, there are 42 ng of DNA in the band at the 10 KB size. Just be sure to include the ul of DNA you loaded in your math.