Daniel:Notebook/HiResChrPaint/2014-6-20
Probe Design (Started 06/17/2014)
Probe Mining
Well, it took a while, but the OligoArray script finished running and...
The Oligo.txt file has nothing in it. Most probes were denied due too high of a Tm. Guess I've got to redo the script but with bigger range for Tms. Sigh...
It would appear that I should have seen this coming. From a brief investigation into Tms of oligos, most oligos of length 60bp have Tms in the 70-100 range, which is way too high given the parameters I gave.
- Run OligoArray2
- java -Xmx4096m -jar /home/djacobse/blastdb_hg38/OligoArray2_1/OligoArray2.jar -i in_caskgene.fas -d hg19.fas -o oligo_caskgene.txt -r rejected_oligo.fas -R caskgene.log -n 30 -l 60 -L 60 -D 1000 -t 35 -T 95 -s 70 -x 70 -p 35 -P 80 -m "CCCC;GGGG;TTTTT;AAAAA" -g 2 &
FISH
I have done some reading while I am waiting for these OligoArray scripts to run, and I may need to increase the formamide concentration for my probes. It occurs to me that I have 60bp probes (genomic annealing), compared with the 32bp published by Beliveau et al. In general, the melting temperature (Tm) of short oligonucleotides increases with the length of nucleotides. Based on this, I took a look, and found this curve from IDT: