Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-15 exp2: Difference between revisions
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>Sam Chiang |
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E.coli IM (43.6ng/uL) 4.59 1.146 - 1.146 - - | E.coli IM (43.6ng/uL) 4.59 1.146 - 1.146 - - | ||
1/10 E.coli IM (4.36ng/uL0 - - 2.293 | 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 | D-C-tRNA (109ng/uL) 2.75 4.128 4.49 4.128 4.49 4.587 | ||
H2O | H2O 92.66 94.726 93.217 94.726 93.217 95.413 | ||
-------------------------------------------------------------------------------------------------------------- | -------------------------------------------------------------------------------------------------------------- | ||
100.00 100.00 100.00 100.00 100.00 100.00 uL | 100.00 100.00 100.00 100.00 100.00 100.00 uL | ||
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'''NOTE: reactions L1, L2 and L3 will be added with De-P-carrier. Reactions L4, L5 and L6 won't.''' | '''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 | *Purify the DAN mixture with EtOH precipitation | ||
**mimic the real precedures to remove the enzyme and buffers from WGA reactions | **To mimic the real precedures to remove the enzyme and buffers from WGA reactions. | ||
* | *Air dry the pellet. Dissolve the pellet in 34uL H2O. | ||
===End-reparing & Phosphorylation=== | ===End-reparing & Phosphorylation=== | ||
Line 94: | Line 94: | ||
===TBE-PAGE validation=== | ===TBE-PAGE validation=== | ||
*Evaluate 5uL of PCR product on TBE-PAGE | |||
*Sample well: 5uL sample + 5uL 0.5X TBE + 5uL 6X loading dye | |||
[[File:sam021710-qPCR 11C-new.jpg|400px]] | [[File:sam021710-qPCR 11C-new.jpg|400px]] | ||
'''L1 L2 L3 L4 L5 L6''' | '''L1 L2 L3 L4 L5 L6''' | ||
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===TBE-PAGE validation=== | ===TBE-PAGE validation=== | ||
*3uL PCR | *3uL PCR products were run on 10-well PAGE (200V, 30min). | ||
**Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer | **Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer | ||
**Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer | **Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer | ||
[[File:sam022210-2nd PCR lib.JPG|400px]] | |||
L1 L2 L3 L4 L5 L6''' | |||
Input DNA 200ng 50ng 10ng 50ng 10ng 0ng | |||
tRNA 300ng 450ng 490ng 450ng 490ng 500ng | |||
De-P-Carrier 450ng 490ng 500ng | |||
Expected size: | |||
Nucleotides Prior-ligation Post-ligation | |||
-------------------------------------------------- | |||
tRNA N/A N/A | |||
Sample (E.coli) 200bp ~300bp | |||
Carrier (Lambda) 100bp ~200bp | |||
==Discussion== | |||
*The the Dephosphorylated carriers are still ligated with adaptor in this tests. | |||
**Sol1: Adjust the dephosphorylation conditions. | |||
**Sol2: Add heat inactivation stet right after End-reparing reaction. | |||
*The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples. | |||
**'''I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.''' | |||
*In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl. |
Latest revision as of 06:06, 3 March 2010
Low-Input DNA libarary construction - Internal marker templates test-1 [edit]
Background[edit]
- 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
- Size-selection prior PCR amplification
Pocedures[edit]
- 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 92.66 94.726 93.217 94.726 93.217 95.413 -------------------------------------------------------------------------------------------------------------- 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
- To mimic the real precedures to remove the enzyme and buffers from WGA reactions.
- Air dry the pellet. Dissolve the pellet in 34uL H2O.
End-reparing & Phosphorylation[edit]
- End-it Kit (EPICENTRE)
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
- Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
- Adding De-P-Carrier into the DNA mixture during QIAquick washing.
- 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
- Elute DNA in 28uL EB
A-tailing[edit]
- Exo-minus klenow DNA pol. kit (Epicentre)
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
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- QIAquick purification. Eluted in 20uL EB.
ligation[edit]
- 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 T4 ligase 2.0 13.0 ------------------------------------ 30.0 123.5/6.5=19
- Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
- QIAquick purification. Eluted in 20uL EB.
Libraray amplification by qPCR[edit]
- 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
- The amplification was stopped at the 11th cycle.
TBE-PAGE validation[edit]
- Evaluate 5uL of PCR product on TBE-PAGE
- Sample well: 5uL sample + 5uL 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[edit]
- 20uL PCR product samples were run on 5-well PAGE (200V, 30min).
- Sample well: 20uL sample + 10uL 6X loading dye + 10uL 0.5X TBE buffer
- Ladder well: 0.5uL low mass ladder + 10uL 6X loading dye + 30uL 0.5X TBE buffer
File:Sam022210-pre size selection -L1-L2-L3.jpg File:Sam022210-post size selection -L1-L2-L3.jpg File:Sam022210-pre size selection -L4-L5-L6-new.jpg File:Sam022210-post size selection -L4-L5-L6.jpg
- DNA was recovered from the gel slice and purified using EtOH purification.
- Air dry the DNA pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.
2nd Libraray amplification by qPCR[edit]
- 2X Fusion mastermix (Enzymatic)
1rxn 6.5rxn H2O 10.8 70.2 DNA 10.0 - 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
- The amplification was stopped at the 7th cycle.
TBE-PAGE validation[edit]
- 3uL PCR products were run on 10-well PAGE (200V, 30min).
- Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE buffer
- Ladder well: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE buffer
File:Sam022210-2nd PCR lib.JPG L1 L2 L3 L4 L5 L6 Input DNA 200ng 50ng 10ng 50ng 10ng 0ng tRNA 300ng 450ng 490ng 450ng 490ng 500ng De-P-Carrier 450ng 490ng 500ng Expected size: Nucleotides Prior-ligation Post-ligation -------------------------------------------------- tRNA N/A N/A Sample (E.coli) 200bp ~300bp Carrier (Lambda) 100bp ~200bp
Discussion[edit]
- The the Dephosphorylated carriers are still ligated with adaptor in this tests.
- Sol1: Adjust the dephosphorylation conditions.
- Sol2: Add heat inactivation stet right after End-reparing reaction.
- The smear (unspecific amplification) is weaker in those samples with lambda DNA carriers. Perhaps the Y-adaptor were comsumed more in those samples.
- I assumed that the overdose of Y-adaptor might be associated with the unspecific amplification.
- In the no lambda carries samples, even the 10ng input-DNA could be successfully made to library using the current protocl.