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='''Solexa sequencing library construction - real samples (human chro MDA product)'''= ==Background== *Sample information **Sample No.10: 1/12.5 (200chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL) **Sample No.12: 1/25 (100chr) 10hr incubation, ~36ng/uL in 20uL. (60ng/uL * 12 uL /20uL) *Only 10uL of each sample were used for this construction (save another 10uL sample as backup). ==TBE-PAGE size selection== *TBE-PAGE condition: 5-well TBU. Run at 200V, 30min **Sample: 10uL sample + 5uL H2O + 5uL 6X loading dye **Ladder: 0.5uL 25bp ladder + 15uL H2O + 5uL 6X loading dye **Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right) **Stained with 3uL SYBR-Gold in 0.5X TBE for 5min *Cut the size 250+/-50bp (200bp~300bp) [[Image:sam102909-TBE-size selection.JPG|350px]] [[Image:sam102909-TBE-size selection-post cutting.JPG|330px]] Left: Pre-cutting Right: Post-cutting ==Blunting== *End-it Kit (EPICENTRE) **Set reactions on cold block 1rxn 2.5rxn DNA 28.0 - 10X blunting buf. 4.0 10.0 dNTP mix 4.0 10.0 Blunting Enzyme 4.0 10.0 ------------------------------------ 40.0 30.0 30/2.5=12 --- DNA 28uL *Mix the reaction by vortexing 5sec. Incubate at RT for 30min. **During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing) *No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB. ==A-tailing== *Exo-Minus Klenow DNA polymerase(EPICENTRE) **Set reactions on cold block *Prepare the fresh dilution of 100mM dATP to 1mM dATP 1rxn 2.5rxns(EPI) DNA 30 - 10X buf. 4 10 (10X Knenow buf.) 1mM dATP 6 15 Klenow enzyme 2 5 ------------------------------------------ 40 30/2.5=12 - DNA 30uL *Mix the reaction by vortexing 5sec. Incubate at 37C for 30min. **During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation) *No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB. ==Ligation== *T4 ligation kit (Enzymatic) **Set reactions on cold block *Adaptor:Insert calculation **Assume that only 25% DNA left after all of previous steps. 360ng*25% =90ng **DNA before ligation: 90ng of 250bp **90/(660x250)x10^6/1000 = 0.54uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 5.4uM ~ 27uM Y-adaptor needed. **I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule. 2.5rxn A-tailed DNA 11 - 2X rapid liagas buf. 15 37.5 20uM Y-adaptor 2 5.0 T4 ligase 2 5.0 --------------------------------- 30 47.5/2.5 = 19uL --- DNA 11 uL *Mix the reaction by vortexing 5sec. Incubate at RT for 15min. *No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB. ==TBU-PAGE size selection== *TBU-PAGE condition: 5-well TBU. Run at 200V, 30min **Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye **Ladder: 0.5uL 25bp ladder + 9uL H2O + 10uL 2X TBU-loading dye **Heat denaturing: 75C 15min -> 1min on cool block **Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right) **Stained with 3uL SYBR-Gold in 0.5X TBE for 5min [[Image:sam103009-TBU.jpg|400px]] [[Image:sam103009-TBU-post cutting.jpg|380px]] Left: Pre-cutting Right: Post-cutting ==Size selected DNA recovery from TBU-PAGE== *Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube. *Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C for 2hr on the shaker. shaking at lowest speed. *Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant on a Nanosep column filter. *Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through. *Perform DNA purification with EtOH/NaOAc method. **Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue. Incubate at -80C for 25min (or longer) **Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant. **Wash the pellet with 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant. **Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
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