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==Troubleshooting== *According to illumina tech support the adapters come double-stranded PE Adapters 5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT {| {{table}} | align="center" style="background:#f0f0f0;"|'''Name in original paper''' | align="center" style="background:#f0f0f0;"|'''Nick-name''' | align="center" style="background:#f0f0f0;"|'''Sequence (5\'->3\')''' | align="center" style="background:#f0f0f0;"|'''Sequence Modification''' | align="center" style="background:#f0f0f0;"|'''Reference''' |- | PCR_F||Sol. Amp. (Forward)||AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] |- | PCR_R||Sol. Amp. (Reverse)||CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] |- |} *so either ligation failed or we lost the samples due to bisulfite conversion ===Troubleshooting #2 02/02/2012=== ===TBU Gel=== 2. Dilute sample 10x (1uL sample + 4 uL of water) so total is 10uL of sample, and add 5 uL of 2x TBE buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 2.5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield. [[File:ZhangLab_2 2012-02-02 15hr 37min.jpg|350px]] ===PCR w Positive Control=== *Used Alice library w N2 Indx 9 as positive control {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 2X iQ Supermix||10||21 |- | 5uM PCR_R_index+PCR_F||0.8||1.68 |- | Template||1||Do not add to template |- | H2O||8.2||17.22 |- | Total||20||39.9 |} *95C 2min *95C 15sec *60C 30sec *72C 30sec *Plate Read *Go to step2 repeat 19 times *72C 10min *15C hold [[File:ZhangLab_2 2012-02-02 15hr 53min.jpg|350px]] [[File:ZhangLab_2 2012-02-02 17hr 38min.jpg|200px]]
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