doles

Hi,

I suppose, the two oligonucleotides have the same lenght (consist of  an identical number of bases). The PB oligo contains an additional sulfur atom, which increases the mass and size of the PB oligo, therefore, it is okay if its band is slightly higher than that of other oligo. (Otherwise, the running of a oligonucleotide can be slightly depended on the nucleotide composition too.)

I also suppose, you should use the two oligos in the same concentration. According to your suppliers and the measurement of NanoDrop, the oligos have the same concentration, but you have observed different contcentrations on gel (based on a dye such as EtBr).  You should decide in which measurement method you trust. Maybe the accurate result based on spectrophotometer (such as NanoDrop and oligo supplier companies usually use this sort of machines to determine the oligo concentration) is disturbed by the sulfur atom. Have you tried to aneal the oligos with those concentrations which you have obsevered on gel?

To sidestep the problem, is double strand adapters for library available for purchase (I know, the cost matters sometimes.)?

In addition, the ligation step may be crucial. Are you sure that the ligation step works well?

Marton Doleschall
human molecular genetics
http://www.kutlab.hu/doleschall.php

Hi,

If your question is current. You can calculate the minimal volume of RE reaction as follows:  Theoretically one unit of DdeI is defined (according to New England Biolabs) as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl. Practically you sould use much more enzyme, I would first try to use 50 U for overnight for 50 µg PhiX174 DNA. 50 U DdeI is  in 5 µl storage volume (in DdeI produced by New England Biolabs). The volume of enzyme must be maximally 10% of the total volume because of star activity, therefore, the minimal volume of the reaction should be 50 µl in this case. Otherwise, you won't  make a mistake, if you use a larger total volume. If you want to use more concentrated DNA for the labelling step, you can extract the DNA with a kit, and then, DNA can be solved again in a smaller volume.

Marton Doleschall, PhD
human molecular genetics
http://www.kutlab.hu/doleschall.php

Thanks madelingirly for the source. I also learnt the bases from this book (a previous edition), I liked it.

It is a very specific question, and I am not really expert, but I guess the same cause (The proteins of homologous recombination system are not expressed in haploid state.).

I hope a real expert will answer you.

Marton

Hi pito,

I was kidding, excuse me. I have tried to imply that European people often are not native English speakers, and their pronunciation sometimes are not correct. (Of course, omitting h and e letters concern only French language.)

Marton