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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== 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
|-
|-
|25mM MnCl2||||||||||0.08ul
|MnCl2/MgCl2||||||||||0.4ul
|-
|-
|RNase III||||||||||0.1ul
|RNase III||||||||||0.1ul
|-
|-
|dH20||||||||||up to 0.83ul
|dH20||||||||||0.33ul
|-
|-
|Total||||||||||2ul
|Total||||||||||2ul
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:::''Incubate @ 37C 5 min''
:::''Incubate @ 37C 5 min''


:::''Column Purify Samples 1-6''




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|-
|-
|5x Ambion PAP Buffer||||||||||0.72ul  
|5x Ambion PAP Buffer||||||||||0.72ul  
|-
|25mM MnCl2||||||||||0.32ul
|-
|-
|ATP (or mix)||||||||||0.25ul   
|ATP (or mix)||||||||||0.25ul   
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|40U/ul Rnase Inhibitor||||||||||0.2ul
|40U/ul Rnase Inhibitor||||||||||0.2ul
|-
|-
|dH2O||||||||||0.31ul
|dH2O||||||||||0.63ul
|-
|-
|}
|}
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:::''Incubate @ 37C 10 min''
:::''Incubate @ 37C 10 min''
:::''Transfer to Ice''
:::''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
  1. No RNase III (5mM MnCl2)/no PAP treatment
  2. RNaseIII (5mM MnCl2)/no PAP treatment
  3. RNaseIII (5mM MgCl2)/no PAP treatment
  4. RNaseIII (10mM MnCl2)/no PAP treatment
  5. RNaseIII (15mM MnCl2)/no PAP treatment
  6. RNaseIII (20mM MnCl2)/no PAP treatment
  7. No RNase III (5mM MnCl2)/PAP (2.5mM MnCl2)
  8. RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2)
  9. RNaseIII (5mM MgCl2)/PAP (2.5mM MgCl2)
  10. RNaseIII (10mM MnCl2)/PAP (5mM MnCl2)
  11. RNaseIII (15mM MnCl2)/PAP (7.5mM MnCl2)
  12. 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]

  1. 1mM ATP:0mM dATP (1:0)
  2. 1mM ATP:0.05mM dATP (1:20)
  3. 1mM ATP:0.1mM dATP (1:10)
  4. 1mM ATP:0.2mM dATP (1:5)
  5. 1mM ATP:1mM dATP (1:1)
  6. 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