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== '''Library Construction: Microwell Amplicons ''' == *We want to try without EtOH precipitation and compare to normally prepared libraries *Cursory research has shown that LMW/HMW is very similar to NEB buffer 4. Additionally, Pol I is active in NEB buffer 4. *I will substitute in this buffer for the Pol I reactions and scale down the reactions *Use 8 amplicons with new, and 8 amplicons with old library preparation *I will do new and Andrew will do old prep *I will also use 2 positive controls with 25 ng template: 1 with new and 1 with old prep '''Pol 1 treatment''' *Start with 1 uL DNA *Add 1.5 uL ALS, 3min *Put on ice and add 1.5 uL NS '''New Prep''' Template 4 uL 10x buffer 4 .7 uL dNTP .35 uL N6(unmodified) .35 uL water .4 uL Pol I .5 uL ---------------------------------- Total 7 uL '''Old Prep''' Template 4 uL 10x buffer 2 2 uL dNTP 1 uL N6(unmodified) 1 uL water 11.5 uL Pol I .5 uL ---------------------------------- Total 20 uL *37 C for 30 min, 65C for 10 min *Go directly to tagmentation for new prep, EtOH for old prep '''Tagmentation''' DNA in water 7 uL HMW buffer 2 uL, 1:50 diluted enzyme 1 uL ---------------------- Total 10 uL *55C for 5 min '''Protease treatment''' *Add 1 uL 1:100 diluted protease *10 min 50C, 20 min 70C '''Klenow''' *Add 1 uL exo- klenow and 1 uL dNTP to each *37C for 15 min, 75 for 20 min '''1st PCR round''' DNA 13 uL KAPA Robust mix 15 uL Orange .6 uL blue .6 uL 50x SYBR .24 uL -------------------------- Total 30 uL *95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8 '''Second round PCR''' *Pause PCR program and add following to each 1st round PCR mix 30 uL KAPA Robust mix 35 uL Adapter 1 2 uL Adapter 2 Barcode 2 uL SYBR Green 1 .56 uL water 30.44 uL ---------------------- Total 100 uL 1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min '''Bead purfication''' *Add 100 uL beads to each, mix, and let sit at RT for 8min *Transfer tubes to magnetic rack and let sit for 5 min *Remove supernatant *2x 80% EtOH wash *Sit for 15 min *Elute in 23 uL Qiagen buffer '''Results''' *The positive controls both worked. However, the new protocol used 7 more cycles, implicating that there is 1000x less DNA. This means that the tagmentation is 1000x less efficient *I want to try 1 more test tomorrow, in which I only add .6 uL of HMW buffer to the tagmentation rxn to make the buffer conc. truly 1x. It seems that higher conc. affect the tagmentation efficiency, and the rxn is very sensitive to buffer conc. *Some of the microwell libraries were positive, though much fainter and most likely have high clonal rates. *For the libraries that Andrew prepared using the EtOH method, none of them worked. I am not sure of the cause since I did not prepare them. But something went wrong with tagmentation since mine seem to have worked. I will have to prepare future samples myself since we are in a crunch. [[File: 11-13-12 pcr.jpg|400px]] [[File: 11-13-12 gel1.jpg|400px]] [[File: 11-13-12 gel2.jpg|400px]]
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