Blue:RNA-Seq Experiments:11052013: Difference between revisions
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>B1lake (Created page with "== Purpose == *Use bioanalyzer to test different fragmentation/poly(A) conditions == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||...") |
>B1lake |
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(9 intermediate revisions by the same user not shown) | |||
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== RNAseIII-Fragmented Library Preparation == | == RNAseIII-Fragmented Library Preparation == | ||
:'''Conditions''' | |||
#No RNase III (5mM MnCl2)/no PAP treatment | |||
#RNaseIII (5mM MnCl2)/no PAP treatment | |||
#RNaseIII (5mM MgCl2)/no PAP treatment | |||
#RNaseIII (10mM MnCl2)/no PAP treatment | |||
#RNaseIII (15mM MnCl2)/no PAP treatment | |||
#RNaseIII (20mM MnCl2)/no PAP treatment | |||
#No RNase III (5mM MnCl2)/PAP (2.5mM MnCl2) | |||
#RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2) | |||
#RNaseIII (5mM MgCl2)/PAP (2.5mM MgCl2) | |||
#RNaseIII (10mM MnCl2)/PAP (5mM MnCl2) | |||
#RNaseIII (15mM MnCl2)/PAP (7.5mM MnCl2) | |||
#RNaseIII (20mM MnCl2)/PAP (10mM MnCl2) | |||
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|RNA||||||||||1.17ul | |RNA||||||||||1.17ul | ||
|- | |- | ||
| | |MnCl2/MgCl2||||||||||0.4ul | ||
|- | |- | ||
|RNase III||||||||||0.1ul | |RNase III||||||||||0.1ul | ||
|- | |- | ||
|dH20|||||||||| | |dH20||||||||||0.33ul | ||
|- | |- | ||
|Total||||||||||2ul | |Total||||||||||2ul | ||
Line 51: | Line 67: | ||
:::''Incubate @ 37C 5 min'' | :::''Incubate @ 37C 5 min'' | ||
:::''Column Purify Samples 1-6'' | |||
Line 62: | Line 79: | ||
|- | |- | ||
|5x Ambion PAP Buffer||||||||||0.72ul | |5x Ambion PAP Buffer||||||||||0.72ul | ||
|- | |- | ||
|ATP (or mix)||||||||||0.25ul | |ATP (or mix)||||||||||0.25ul | ||
Line 71: | Line 86: | ||
|40U/ul Rnase Inhibitor||||||||||0.2ul | |40U/ul Rnase Inhibitor||||||||||0.2ul | ||
|- | |- | ||
|dH2O||||||||||0. | |dH2O||||||||||0.63ul | ||
|- | |- | ||
|} | |} | ||
Line 77: | Line 92: | ||
:::''Incubate @ 37C 10 min'' | :::''Incubate @ 37C 10 min'' | ||
:::'' | :::''Column Purify Samples 7-12'' | ||
[[File:2100_expert_Eukaryote_Total_RNA_Pico_DE72903057_2013-11-06_12-24-52.pdf]] | |||
Results: | |||
*RNaseIII fragmentation is very efficient (fragments centered on about 75-100nt) and doesn't change with increasing MnCl2 | |||
*PAP is not fully complete, and smear is quite high. Seems to decrease in length with increasing MnCl2 | |||
*Substitution of MnCl2 for MgCl2 shows similar smear pattern as unfragmented - indicating MnCl2 works better for RNaseIII | |||
== Testing dATP on PAP Activity == | |||
#1mM ATP:0mM dATP (1:0) | |||
#1mM ATP:0.05mM dATP (1:20) | |||
#1mM ATP:0.1mM dATP (1:10) | |||
#1mM ATP:0.2mM dATP (1:5) | |||
#1mM ATP:1mM dATP (1:1) | |||
#0mM ATP:1mM dATP (0:1) | |||
1ul 10uM TSO | |||
1ul 5x ePAP buffer | |||
0.5ul 25mM MnCl2 | |||
0.5ul ATP/dATP | |||
1.0 ul 0.4U/ul ePAP | |||
1ul dH2O | |||
[[File:11052013_gel1.jpg]] | |||
Results: dATP was able to limit ePAP activity to a small degree when added 1:1 with ATP |
Latest revision as of 00:17, 7 November 2013
Purpose[edit]
- Use bioanalyzer to test different fragmentation/poly(A) conditions
RNAseIII-Fragmented Library Preparation[edit]
- Conditions
- No RNase III (5mM MnCl2)/no PAP treatment
- RNaseIII (5mM MnCl2)/no PAP treatment
- RNaseIII (5mM MgCl2)/no PAP treatment
- RNaseIII (10mM MnCl2)/no PAP treatment
- RNaseIII (15mM MnCl2)/no PAP treatment
- RNaseIII (20mM MnCl2)/no PAP treatment
- No RNase III (5mM MnCl2)/PAP (2.5mM MnCl2)
- RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2)
- RNaseIII (5mM MgCl2)/PAP (2.5mM MgCl2)
- RNaseIII (10mM MnCl2)/PAP (5mM MnCl2)
- RNaseIII (15mM MnCl2)/PAP (7.5mM MnCl2)
- RNaseIII (20mM MnCl2)/PAP (10mM MnCl2)
- RNA
Volume UHRR 10ng/ul 1ul 5x Ambion PAP buffer 0.08ul dH20 0.09ul Total 1.17ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- RNA FRAGMENTATION
Volume RNA 1.17ul MnCl2/MgCl2 0.4ul RNase III 0.1ul dH20 0.33ul Total 2ul
- Incubate @ 37C 5 min
- Column Purify Samples 1-6
- 3' Tailing
Volume fRNA 2ul 5x Ambion PAP Buffer 0.72ul ATP (or mix) 0.25ul 2U/ul PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.63ul
- Incubate @ 37C 10 min
- Column Purify Samples 7-12
File:2100 expert Eukaryote Total RNA Pico DE72903057 2013-11-06 12-24-52.pdf
Results:
- RNaseIII fragmentation is very efficient (fragments centered on about 75-100nt) and doesn't change with increasing MnCl2
- PAP is not fully complete, and smear is quite high. Seems to decrease in length with increasing MnCl2
- Substitution of MnCl2 for MgCl2 shows similar smear pattern as unfragmented - indicating MnCl2 works better for RNaseIII
Testing dATP on PAP Activity[edit]
- 1mM ATP:0mM dATP (1:0)
- 1mM ATP:0.05mM dATP (1:20)
- 1mM ATP:0.1mM dATP (1:10)
- 1mM ATP:0.2mM dATP (1:5)
- 1mM ATP:1mM dATP (1:1)
- 0mM ATP:1mM dATP (0:1)
1ul 10uM TSO
1ul 5x ePAP buffer
0.5ul 25mM MnCl2
0.5ul ATP/dATP
1.0 ul 0.4U/ul ePAP
1ul dH2O
Results: dATP was able to limit ePAP activity to a small degree when added 1:1 with ATP