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='''Low-input DNA Solexa seq. library construction test using dUTP-carrier'''= ==Background== *It has been proved that dUTP-carrier can be digested by USEr enzyme and S1 endonuclease. *I am testing if dUTP-carrier can protect low-input DNA in library construction. **The low-input DNA sample were obtained from the NlaIII digested Jurket DNA (25ng/uL) from 10/23/'09 exp. ===Carrier preparation=== *EtOH/NaOAC purify the dUTP-PCR product made on 11/17/'09 **100uL dUTP-PCR product + 250uL 100%EtOH + 10uL 3M NaOAc + 1.5uL Glycoblue -> -80C precipitation -> 750uL 75% EtOH washing **E1U-e: 386.52 ng/uL 1ug=2.58uL **E3U-e: 181.06 ng/uL 1ug=5.52uL ===Sample preparation=== *NlaIII digested Jurket DNA 500ng/20uL=25ng/uL 50ng-Carrier 100ng-Carrier 200ng-Carrier DNA sample 2.00uL 4.00uL 8.00uL E1U-e(1ug) 2.58 2.58 2.58 ------ 1ug dUTP-dsDNA carrier H2O 23.42 21.42 17.42 -------------------------------------------------------- 28.00 28.0 28.00 50ng-Carrier 100ng-Carrier 200ng-Carrier DNA 2.00uL 4.00uL 8.00uL E3U-e(1ug) 5.52 5.52 5.52 ------ 1ug dUTP-dsDNA carrier H2O 20.48 18.48 14.48 -------------------------------------------------------- 28.00 28.00 28.0 ===Blunting=== *End-it Kit (EPICENTRE) **Set reactions on cold block 1rxn 7rxn DNA 28.0 - 10X blunting buf. 4.0 28.0 dNTP mix 4.0 28.0 Blunting Enzyme 4.0 28.0 ------------------------------------ 40.0 84.0 84/7=12 --- DNA 28uL Add the DNA pol.I (E.coli) 1uL on the top of reaction *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. '''NOTE: The reaction B3-2 and B3-3 maybe mis-labeled in this step.''' ===A-tailing=== *Exo-Minus Klenow DNA polymerase(EPICENTRE) **Set reactions on cold block *Prepare the fresh dilution of 100mM dATP to 1mM dATP 1rxn 7rxns DNA 30 - 10X Knenow buf. 4 28 1mM dATP 6 42 Klenow enzyme 2 14 ------------------------------------------ 40 84/7=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 50% DNA left after all of previous steps (no sample size-selpection). (1ug + X ng)/2=500ng (X=sample amount -> ignored in the calculation) **DNA before ligation: 500ng of 250bp **500/(660x250)x10^6/1000 = 3.03uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 30.3uM ~ 151.5uM Y-adaptor needed. **I decided to use 3uL of 20uM Y-adaptor which is in between 10:1 and 50:1 rule. 7rxn A-tailed DNA 11 - 2X rapid liagas buf. 15 105.0 20uM Y-adaptor 3 21.0 T4 ligase 2 14.0 ---------------------------------- 30 140.0/7 = 20uL --- DNA 10uL '''NOTE: In order to increase production, I decided to add all of the DNA (12uL) eluted from previous step.''' *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 0.5X TBE + 10uL 2X TBU-loading dye **Heat denaturing: 75C 15min -> on cool block (>1min) **Stained with 3uL SYBR-Gold in 0.5X TBE for 5min [[Image:sam111909-tbu gel 1.jpg|400px]] [[Image:sam111909-tbu gel 1-post cut.jpg|350px]] Left: Pre-cutting Right: Post-cutting [[Image:sam111909-tbu gel 3.jpg|350px]] [[Image:sam111909-tbu gel 3-post cut.jpg|360px]] Left: Pre-cutting Right: Post-cutting ===Recovery of size selected DNA=== *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 shaker (>2hr or overnight). shaking at the lowest speed. *Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant onto 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 750uL 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. ===qPCR amplification=== *Only use half of amount (10uL)of ligation product for qPCR amplification. *Solexa lib. templates: **Left to right: L1-1, L1-2, L1-3, L3-1, L3-2, L3-3 *Primers - from Dr. Zhang **(Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM) **(Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM) Master mix 6.5 rxn H2O 31.6 205.4 Template 10.0 - Solexa_PCR_upH (10uM) 4.0 26.0 Solexa_PCR_PE_lo_H (10uM) 4.0 26.0 SYBR 50X 0.4 2.6 2X Fusion enzyme mix(Enzymatics) 50.0 325.0 ----------------------------------------------- 100.0 585.0/6.5=90 --- 10uL template *Using two annealing temp program for amplification **Use 5 cycles for the first annealing temp(64C) - step5. **Use 16 cycles for the second annealing temp(72C) - step9. *Immediately transfer 50uL of reaction into a fresh PCR tube for an extra cycle amplification in Chromo4. *Terminate/pause the reaction at annealing step when it's closing to saturation stage. [[Image:sam112009-solexa lib qPCR step5.jpg|600px]] [[Image:sam112009-solexa lib qPCR step9.jpg|600px]] The reaction were terminated at the 9th and 10th cycles [[File:sam112309-low input DNA library.jpg|400px]] '''Carrier size: L1(260 bp), L3(245 bp)''' *The result suggested that the library from 50ng input-DNA can barely be seen on gel. '''50ng probably is the minimum of lowest input-DNA for libaray consturction'''. *A problem of undigested carrier remained in the DNA sample. **Based on the size it seems these carrier are also ligated with adaptors (L1: 260->360bp; L3: 245->345bp) **However these carriers are from know sequnce of E.coli gDNA. They could be easily removed from sequnce result computationally.
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