Hi Chandan,

Can you explain the precise protocol you used, particularly with regard to preparation of the column, application of the sample and the elution spins?  This is usually the source of issues that can lead to nucleotides running through and being collected instead of being retained within the resin while the longer fragments run through.

Best wishes.

Hi Robin

I have used illustra Micro-spin G50 columns. The protocol is almost the same they mention in their product usage manual. My product size is ~80bp.

1.Give a spin of 800RCF(G) for 2min to the spin columns to drain off the buffer.

2.Place the column in t a fresh Eppendorf tube for collection of your sample.

3. Carefully aliquot the labeled sample (I use 40uL) onto the columns.( I try my best to place the samples in the centre of the column). Immediately spin at 800RCF for another 2 min .

4. 2uL of the Sample collected is run on the TBE-Urea gel for visualization.

I hope I am not missing anything.

Thanks

Chandan.

with sephadex columns

Pablo

Hi Chandan,

let's assume for the moment that the columns are working exactly as intended.  In gel filtration (whether large column or spin column) the various peaks will travel through the column as a factor of time against the separation/exclusion characteristics of the resin - large fragments first in the void, then smaller fragments that can travel through the matrix volume as well.  For whatever reason at the moment your product and the unincorporated nucleotides are travelling through together.  As an initial experiment, instead of following the exact protocol, could you consider eluting in "temporal" aliquots : instead of spinning for 2'00, instead spin to collecting tube 1 for 30s, then collecting tube 2 for 30s, then collecting tube 3 for 30s and so on - then run the samples out?  This should hopefully separate the desired product from the nucleotides, and may also give you insights as to an optimal elution spin time.

Good luck with your experiment!

Dear Robin,

I did exactly as you have mentioned. It worked. Initial fraction spun for the short time gave me a better yield of desired bands than the latter fractions.

Thank you :)

Dear Pablo, 

Illustra columns indeed has sephadex. Thank you for the response. I could figure out the solution.

Hi Chandan, that's brilliant to hear - I am really pleased that this idea worked!  Good luck with the rest of your project!

Thanks Robin. Good day.

You can try using Sephadex G10 or G25 columns.