Today I did two reactions that labeled the ends of digested chromosomal DNA with biotin (one of EcoRI-digested DNA and one of XhoI-digested DNA). The next step is to clean up the DNA, to get rid of the Klenow polymerase and the EcoRI/XhoI and the unincorporated nucleotides. it's especially important to get rid of ALL of the unincorporated biotin-dUTP, because this will otherwise bind to the streptavidin-coated beads and prevent the biotinylated DNA from binding to them.
Because I want to wash the DNA well to get rid of the biotin-dUTP, a column cleanup is best. We have new cheap cleanup columns from a company called Epoch, to replace the relatively expensive Sigma genelute columns we've been using. (These in turn replaced very expensive columns from Qiagen.) What makes the Epoch columns such good value is that instead of charging for little bottles of salty water like the other companies (their 'secret sauce' buffers), Epoch just provides the recipes so users can make their own buffers.
The RA had already tested the new columns with a PCR cleanup, but I needed to test them with large fragments of chromosomal DNA, because DNA fragments bigger than 10-20 kb tend to stick to this kind of column. Bottom line: both columns release almost all the DNA fragments smaller than 20 kb.
I also wanted to compare the overall recovery of DNA from the columns, especially if they were heavily loaded with a lot of DNA (their stated binding capacities were either 10 µg or 20 µg, depending on which document I read). But my DNA wasn't as concentrated as I thought, so the most DNA I put on a column was probably about 14 µg. Recoveries were good, >80% even with more than 10 µg on the column.
So now I have about 35 µg of biotin-tagged EcoRI-cut chromosomal DNA, and about 25 µg of biotin-tagged XhoI-cut chromosomal DNA. The next step is to measure binding of this DNA to the streptavidin-coated beads. I can do this accurately now that the RA has shown me how to use Picogreen to measure very low DNA concentrations, and I've checked that beads don't interfere with these measurements. I wasn't sure if I could put samples containing beads onto the NanoDrop spec, but I just read their explanation of how it works and I don't see any problem. Maybe I'll email their Customer Service people just to be sure, as the NanoDrop we use belongs to the lab next door.
Friday Fabulous Flower - Geranium?
2 days ago in The Phytophactor