Sam:LabNotes/Human-Chip-bisulfite-Seq/2009-11-19
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Low-input DNA Solexa seq. library construction test using dUTP-carrier[edit]
Background[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[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 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
File:Sam111909-tbu gel 1.jpg File:Sam111909-tbu gel 1-post cut.jpg Left: Pre-cutting Right: Post-cutting File:Sam111909-tbu gel 3.jpg File:Sam111909-tbu gel 3-post cut.jpg Left: Pre-cutting Right: Post-cutting
Recovery of size selected DNA[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 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[edit]
- 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.
File:Sam112009-solexa lib qPCR step5.jpg File:Sam112009-solexa lib qPCR step9.jpg The reaction were terminated at the 9th and 10th cycles
File:Sam112309-low input DNA library.jpg 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.