ekg/r2dm

R2 transposon in Drosophila melanogaster

R2Dm

a target primed reverse transcription transposon

Reference sequence is 3607bp long.

 % oligotm $(samtools faidx r2dm.fa r2dm:0-3607 | tail -n+2 | perl -pe 's/\n//')                                                                                                                 ~/r2dm (master) oog
101.095285

TOPO cloning primers

Putative primers.

Forward:

samtools faidx r2dm.fa r2dm:1-21 | tail -1 >primer.5p
# TTGGGGATCATGGGGTATTTG

Reverse:

# design chops off polyA tail and one C
./revcomp $(samtools faidx r2dm.fa r2dm:3558-3585 | tail -1 | tail -c 23 | head -c 21) >primer.3p
# GATCGCGGAGGTATGGAAAT

(Un-chopped version ordered, but may not use initially.)

Secondary primers, somewhere in the middle (1632 bp):

echo AAGGCATTTGATTCTCTATCACATG >primer.mid.5p
./revcomp $(cat ./primer.mid.5p ) >primer.mid.3p

Tm are within 5C. Oligtm is in primer3.

oligotm $(cat primer.5p)
# 63.738920
oligotm $(cat primer.3p)
# 62.018127
oligotm $(cat primer.mid.5p)
# 60.706056

Note that the two-step PCR would yield intermediate products of 1632bp and 1953bp. A full-length product should be 3585bp.