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==Qiaquick Column Purification== ===Purify with one column per reaction=== *In 1.5mL tube, add 6x volume of PB buffer to each tube with PCR product and mix (600ul to 100 ul of PCR product) *Transfer 700ul of mixture to each qiaquick column *spin at 14,000rpm for 1". Discard supernatant *Add 750ul wash buffer to each column *spin at 14,000rpm for 1". Discard supernatant *repeat wash with wash buffer *transfer column to 1.5mL lowbind tube *Add 50ul 55C EB buffer to each column and incubate in 37C for 5" *Elute DNA by spinning at 14,000 rpm for 1 min. *Pool the purified amplicon into the same tube (total volume ~100ul). *Measure DNA concentration by Nanadrop and calculate the total yield (dsDNA). *Dilute purified 1st round amplicon to 10nM with TE buffer. ===Nanodrop=== [[File:12132013_GP1_V4_ND1.JPG|300px|GP1_V4]] [[File:12132013_GP1_V6_ND1.JPG|300px|GP1_V6]] *Left:GP1_V4 Right:GP1_V6 *GP1_V4:10ng/ul, 112.56nM *GP1_V6:9.9ng/ul, 111.44nM *Keep 1ul for Gel quantification ===Dilute probes to 10nM=== *GP1_V4: Add 0.85ul 112.56nM probes to 8.75ul EBuffer *GP1_V6: Add 0.86ul 111.44nM probes to 8.74ul EBuffer
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