Sam:LabNotes/Human/2009-10-29
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Solexa sequencing library construction - real samples (human chro MDA product)[edit]
Background[edit]
- 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[edit]
- 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)
File:Sam102909-TBE-size selection.JPG File:Sam102909-TBE-size selection-post cutting.JPG Left: Pre-cutting Right: Post-cutting
Blunting[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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
File:Sam103009-TBU.jpg File:Sam103009-TBU-post cutting.jpg Left: Pre-cutting Right: Post-cutting
Size selected DNA recovery from TBU-PAGE[edit]
- 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.