Important: Following the PCR, do not open PCR tubes in
same room where iCLIP library preparations (i.e., all steps
before step 8 of Subheading3.11) were carried out. All
post-PCR analysis and storage needs to be carried out in
separate room to avoid amplicon contamination.- Assemble the XCell III gel system according to manufacturer’s
instructions using a 6% TBE gel and TBE running buffer. - Add 2μLof6loading dye to each PCR sample and 10μLof
1:30 low molecular weight marker. Load 12μL of samples/
ladder into gel wells. - Run gel for 30 min at 180 V
- Remove gel from cassette and stain for 5 min in SYBR Safe in
TBE buffer. Visualize using appropriate imager (Fig.4a). The
P5/P3 primers account for 128 nt of PCR product. Appropri-
ate PCR band sizes are therefore:
Fig. 4cDNA library PCRs. (a) Example of final library PCR (steps 13– 17 of Subheading3.11) showing low,
medium, and high bands. Both a low RNase sample and a no UV control sample are shown. (b) Example of a
cDNA library amplified with different PCR cycle numbers.Red boxesindicate secondary spurious products that
migrate at a higher molecular weight to the expected products
446 Christopher R. Sibley