Sam:LabNotes/Human-Chip-bisulfite-Seq/2010-2-27: Difference between revisions
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>Sam Chiang (Created page with '='''Low-Input DNA libarary construction - Internal marker templates test-1 '''= ==Background== *Test low-input DNA libaray conscturction using internal marker at 200ng, 50ng, 10…') |
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='''Low-Input DNA libarary construction - Internal marker templates test- | ='''Low-Input DNA libarary construction - Internal marker templates test-2'''= | ||
==Background== | ==Background== | ||
* | *Repeat the low-input DNA libaray conscturction with following changes: | ||
**Add inactivation step following End-repaining. | |||
** | **Try two more conditions of De-P-Carriers (carriers made by SAP 60min or ACP 60min) | ||
**Change the order of size-selection. '''The size-selection will be performed prior PCR amplification.''' | |||
** | |||
** | |||
==Pocedures== | ==Pocedures== | ||
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**DNAse digested-cleaned (D-C) tRNA 109 ng/uL | **DNAse digested-cleaned (D-C) tRNA 109 ng/uL | ||
Sample name''' | Sample name''' B1 B2 B3 B4 B5 B6 B7 B8''' | ||
200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM | 200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM 0ng IM 0ng IM | ||
300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA | 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA 500ng tRNA 500ng tRNA | ||
----------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------- | ||
E.coli IM (43.6ng/uL) 4.59 1. | E.coli IM (43.6ng/uL) 4.59 1.15 - 1.15 - - - - | ||
1/10 E.coli IM (4.36ng/uL0 - - 2. | 1/10 E.coli IM (4.36ng/uL0 - - 2.29 - 2.29 - - - | ||
D-C-tRNA (109ng/uL) | D-C-tRNA (109ng/uL) 3.85 5.77 6.28 5.77 6.28 6.41 6.41 6.41 | ||
H2O | H2O 91.56 93.08 91.43 93.08 91.43 93.59 93.59 93.59 | ||
-------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------- | ||
100.00 100.00 100.00 100.00 100.00 100.00 uL | 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 uL | ||
'''NOTE: reactions | '''NOTE: reactions B1, B2 and B3 will be added with De-P-carrier. Reactions B4 - B8 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 | **mimic the real precedures to remove the enzyme and buffers from WGA reactions | ||
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*End-it Kit (EPICENTRE) | *End-it Kit (EPICENTRE) | ||
1rxn | 1rxn 8.5rxn | ||
DNA+H2O 34.0 - | DNA+H2O 34.0 - | ||
10X buf. 5.0 | 10X buf. 5.0 42.5 | ||
2.5mM dNTP 5.0 | 2.5mM dNTP 5.0 42.5 | ||
10mM ATP 5.0 | 10mM ATP 5.0 42.5 | ||
Enzyme 1.0 | Enzyme 1.0 8.5 | ||
------------------------------------ | ------------------------------------ | ||
50.0 | 50.0 136.0 136/8.5=16 | ||
*Mix the reaction by vortexing 5sec. Incubate at RT for 45min. | *Mix the reaction by vortexing 5sec. Incubate at RT for 45min. | ||
*Adding De-P-Carrier into the DNA mixture during QIAquick washing. | *Adding De-P-Carrier into the DNA mixture during QIAquick washing. | ||
** | **Concentration of De-P-Carrier 1ug/30uL = 33.3ng/uL | ||
**450ng for | **'''S30''': 450ng for B4 reaction: 450/33.3=13.5uL | ||
** | **'''S30''': 490ng for B5 reaction: 490/33.3=14.7uL | ||
**500ng for | **'''S30''' :500ng for B6 reaction: 500/33.3=15.0uL | ||
**'''S60''' :500ng for B7 reaction: 500/33.3=15.0uL | |||
**'''A60''' :500ng for B8 reaction: 500/33.3=15.0uL | |||
*Elute DNA in 28uL EB | *Elute DNA in 28uL EB | ||
Line 56: | Line 55: | ||
*Exo-minus klenow DNA pol. kit (Epicentre) | *Exo-minus klenow DNA pol. kit (Epicentre) | ||
1rxn | 1rxn 8.5rxn | ||
DNA+H2O 28.0 - | DNA+H2O 28.0 - | ||
10X buf. 4.0 | 10X buf. 4.0 34.0 | ||
1mM dATP 6.0 | 1mM dATP 6.0 51.0 | ||
Enzyme 2.0 | Enzyme 2.0 17.0 | ||
------------------------------------ | ------------------------------------ | ||
40.0 | 40.0 102.0 102/8.5=12 | ||
*Mix the reaction by vortexing 5sec. Incubate at 37C for 30min. | *Mix the reaction by vortexing 5sec. Incubate at 37C for 30min. | ||
*QIAquick purification. Eluted in 20uL EB. | *QIAquick purification. Eluted in 20uL EB. | ||
Revision as of 08:39, 28 February 2010
Low-Input DNA libarary construction - Internal marker templates test-2
Background
- Repeat the low-input DNA libaray conscturction with following changes:
- Add inactivation step following End-repaining.
- Try two more conditions of De-P-Carriers (carriers made by SAP 60min or ACP 60min)
- Change the order of size-selection. The size-selection will be performed prior PCR amplification.
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 B1 B2 B3 B4 B5 B6 B7 B8 200ng IM 50ng IM 10ng IM 50ng IM 10ng IM 0ng IM 0ng IM 0ng IM 300ng tRNA 450ng tRNA 490ng tRNA 450ng tRNA 490ng tRNA 500ng tRNA 500ng tRNA 500ng tRNA --------------------------------------------------------------------------------------------------------------------------------------------- E.coli IM (43.6ng/uL) 4.59 1.15 - 1.15 - - - - 1/10 E.coli IM (4.36ng/uL0 - - 2.29 - 2.29 - - - D-C-tRNA (109ng/uL) 3.85 5.77 6.28 5.77 6.28 6.41 6.41 6.41 H2O 91.56 93.08 91.43 93.08 91.43 93.59 93.59 93.59 --------------------------------------------------------------------------------------------------------------------------------------------- 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 uL NOTE: reactions B1, B2 and B3 will be added with De-P-carrier. Reactions B4 - B8 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. Air dry the pellet. Dissolve the pellet in 34uL H2O.
End-reparing & Phosphorylation
- End-it Kit (EPICENTRE)
1rxn 8.5rxn DNA+H2O 34.0 - 10X buf. 5.0 42.5 2.5mM dNTP 5.0 42.5 10mM ATP 5.0 42.5 Enzyme 1.0 8.5 ------------------------------------ 50.0 136.0 136/8.5=16
- Mix the reaction by vortexing 5sec. Incubate at RT for 45min.
- Adding De-P-Carrier into the DNA mixture during QIAquick washing.
- Concentration of De-P-Carrier 1ug/30uL = 33.3ng/uL
- S30: 450ng for B4 reaction: 450/33.3=13.5uL
- S30: 490ng for B5 reaction: 490/33.3=14.7uL
- S30 :500ng for B6 reaction: 500/33.3=15.0uL
- S60 :500ng for B7 reaction: 500/33.3=15.0uL
- A60 :500ng for B8 reaction: 500/33.3=15.0uL
- Elute DNA in 28uL EB
A-tailing
- Exo-minus klenow DNA pol. kit (Epicentre)
1rxn 8.5rxn DNA+H2O 28.0 - 10X buf. 4.0 34.0 1mM dATP 6.0 51.0 Enzyme 2.0 17.0 ------------------------------------ 40.0 102.0 102/8.5=12
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
- QIAquick purification. Eluted in 20uL EB.