Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-15 exp2
Jump to navigation
Jump to search
Low-Input DNA libarary construction - Internal marker templates
Background
- Test low-input DNA libaray conscturction using internal marker at 200ng, 50ng, 10ng at 0ng, with or without De-P-carrier.
- Goals:
- Test the newly synthesized adaptors and primers by myself.
- Test Celso's protocol (with new End-repairing recipe).
- Get the reference position of post-ligation reactions from internal marker template.
- Compare the effect of adding De-P-carrier or not on library construction.
- Test the effect of contamination by De-P-carrier
Pocedures
- Prepare the following samples
- E.coli internal marker (IM) (200bp): 43.6ng/uL
- 1/10 dilutted E.coli internal marker (IM) (200bp): 4.36ng/uL
- DNAse digested-cleaned (D-C) tRNA 109 ng/uL
Sample name L1 L2 L3 L4 L5 L6 200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA ----------------------------------------------------------------------------------------------------------------- E.coli IM (43.6ng/uL) 4.59 1.146 - 1.146 - - 1/10 E.coli IM (4.36ng/uL0 - - 2.293 2.293 - D-C-tRNA (109ng/uL) 2.75 4.128 4.49 4.128 4.49 4.587 H2O 42.66 44.726 43.217 44.726 43.217 45.413 -------------------------------------------------------------------------------------------------------------- 100.00 100.00 100.00 100.00 100.00 100.00 uL NOTE: reactions L1, L2 and L3 will be added with De-P-carrier. Reactions L4, L5 and L6 won't.
Purify the DAN mixture with EtOH precipitation
- (mimic the real precedures to remove the enzyme and buffers from WGA reactions)
- Each sample (PCR product) has 100uL volume
- In a non-sticky 1.5-mL tube. Add 10uL 3M NaOAc, 250uL 100% EtOH, 1.5uL Glycoblue and 100uL DNA samples. Vortex 5sec.
- Incubation at 80C for 25min. Pelleting at 10,000xg 4C for 25min. Remove supernatant.
- Washing with 750uL 75%EtOH. Pelleting at 10,000xg 4C for 5min. Remove supernatant.
- Air dry the pellet. Dissolve the pellet in 34uL H2O.
End-reparing & Phosphorylation
- End-it Kit (EPICENTRE)
- Set up reactions on cold block
1rxn 6.5rxn DNA+H2O 34.0 - 10X buf. 5.0 32.5 2.5mM dNTP 5.0 32.5 10mM ATP 5.0 32.5 Enzyme 1.0 6.5 ------------------------------------ 50.0 104.0 104/6.5=16 --- DNA 34uL
- Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
- 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 immediately.
- After 45min incubation, add the product (50uL) into 1.5-mL non-sticky tube containg 250uL PB (QIAquick buffer)
- Adding De-P-Carrier into the DNA mixture
- Use De-P-Carrier "3"(100bp): 22uL*40ng/uL/30uL = 30ng/uL
- 450ng for L4 reaction: 450/30=15.0uL
- 490ng for L5 reaction: 490/30=16.3uL
- 500ng for L6 reaction: 500/30=16.6uL
(Vortex the DNA mixture and store the sample in -20C)
- Elute DNA in pre-warmed PB (20 + 8uL)
A-tailing
- Exo-minus klenow DNA pol. kit (Epicentre)
- Dilute the 100mM dATP to 1mM dATP
- Set up reactions on cold block
1rxn 6.5rxn DNA+H2O 28.0 - 10X buf. 4.0 26.0 1mM dATP 6.0 39.0 Enzyme 2.0 13.0 ------------------------------------ 40.0 78.0 78/6.5=12 --- DNA 28uL
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min (Program "A-tailing").
- 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 immediately. Eluted in pre-warmed EB (10 + 10uL).
- Speed vac the sample from 20uL to 11uL (around 10min at default speed).
- NOTE: During the A-tailing step. Prepare the Y-adaptor annealing.
- Preparign 20uM Y-adaptor mix
100uM Sam-PE_t 20uL 100uM Sam-PE_b 20uL 10X T4 ligase buf. 10uL H2O 50uL ------------------------ 100uL 94C 3min -> cool donw to 20C at the rate of 0.1/sec using RAMP function
TA ligation
- T4 ligation kit (Enzymatic)
1rxn 6.5rxn DNA+H2O 11.0 - 2X rapid buf. 15.0 97.5 20uM Y-adaptor 2.0 13.0 --- newly synthesized Sam-PE-adaptor (20uM) T4 ligase 2.0 13.0 ------------------------------------ 30.0 123.5/6.5=19 --- DNA 11uL
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (library amplification by qPCR).
- No inactivation needed, but purify the A-tailed DNA with QIAquick column immediately. Eluted in pre-warmed EB (10+10uL).
Libraray amplification by qPCR
- 2X Fusion mastermix (Enzymatic)
1rxn 6.5rxn H2O 0.8 5.2 DNA 20.0 - PCR_f (10uM) 2.0 13.0 PCR_r (10uM) 2.0 13.0 SYBR 50X 0.2 1.3 2X Fusion enzyme 25.0 162.5 ------------------------------------ 50.0 195.0/6.5=30 --- DNA 20uL (Use all of ligation reaction from the last step)
- Monitor the amplification curves and stop the reaction before reaching the plateau.
- The amplification was stopped at the 11th cycle.
- NOTE: Since the PCR amplification was performed on the non-size selected template, there are lots of noise interfere the correct timing to stop the amplification.
TBE-PAGE validation
- Use 5uL of amplified product for validation on 10-well TBE-PAGE
- Sample loading: 5uL sample + 5uL 0.5X TBE + 5uL 6X loading dye
- Ladder loading: 0.5 low mass ladder + 10uL 0.5X TBE + 5uL 6X loading dye
File:Sam021710-qPCR 11C-new.jpg L1 L2 L3 L4 L5 L6 200ng DNA 50ng DNA 10ng DNA 50ng DNA 10ng DNA 0ng DNA 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA 450ng Carrier 490ng Carrier 500ng Carrier
- DNA size(200bp), carrier size(100bp), Y-adaptor(PE) size(100bp)
- All amplicons showed smear since the templates were non-size selected DNA
- Although the theoretically only adaptor ligated DNA should be amplified, the results showed overall unspecific amplification especially in carrier-added tests (L4, L5 and L6).
- It is also possible that the over-amplification cause these unspecific amplification (The reaction was stopped at 11th cycle for the current test). However, insufficient amplification won't get enough product for low-template DNA reaction such as starting DNA< 10ng.
- The non-carrier test (L1, L2 and L3) showed potential targets band at expected size (~300bp) with gradient intensity. Even the 10ng DNA test (L3) can show the band with 5uL amplified PCR amplicons.
- The carrier-added tests showed weaker potential targets band.
- It is possible the carrier was ligated with Y-adaptors, which generate the band at size of ~200bp.
- I should add the Heat-inactivation after I did the End-repairing reaction.
Size selection
- The potential target bands were size selected on 5-well TBE-PAGE (x2).
- Sample loading: 20uL sample + 5uL 0.5X TBE + 5uL 6X loading dye
- Ladder loading: 0.5 low mass ladder + 20uL 0.5X TBE + 5uL 6X loading dye
- Load ladder between each samples. Run at 200V for 30min.
File:Sam022210-pre size selection -1.jpg