Contributed by Riddhish Shah, Hueley Lab

• Gel Preparations:

1. Assembly for gel casting: Take clean plates and the spacers. Snuggly tighten them to make casting module. Fit it on the stand without the rubber spacer on bottom. Add some water to make sure water is not leaking out at the bottom. Keep the comb handy. Drain off the water by inverting the gel cast. Few droplets sticking to glass plates don’t interfere with gel.

2. Gel solution preparation and casting:

Water distilled 50 ml

30:2 acralamide: bis  8 ml

10X TBE  1.5 ml

10% APS  600 ul (0.0600 gm in 600 ul)

Temed 60 ul

Add temed at last. Mix well. Now gel is ready to pour.

Take 25ml pipette and try to pour the gel on the side of cast near spacers. So it trickles on the side and fills from bottom without any air bubbles trapped inside. Fill it up to the top. Take comb and insert from one side keeping angle so that each tooth of comb can be inserted one by one in Gel without trapping any air bubble. The gel should polymerize in half and hour and ready to use. Make sure its not leaking from anywhere when you leave it to polymerize.

• Oligoneucleotide Labeling.

We want our final labeled product to be 4.8 pm/ul so dilute initial single stranded oligoneculeotide at 480 pm/ul. Now we need to anneal them.

100 ul of annealing buffer ( 10mm Tris-HCl, Ph 7.5, 20mm NaCl)

1 ul or each primer (480 pm of each of them)

Heat it for 95* for 5 min on heat block. Leave the block at RT afterwards.

Store at