Brandon:LabNotes/Project1/2013-5-10

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AMPURE beads testing Experiment continued 6/4[edit]

  • test AMPURE beads for DNA, RNA and Zymo purification and quantify loss. Previous assays have shown great sample loss, thus need to repeat.


  • Using pure DNA samples from which IVT RNA was generated. Will use AMPURE beads labeled for DNA, AMPURE beads labeled for RNA and Zymo Kit as control.


  • also will reduce drying time to 3 minutes, since that causes higher stress on the sample.


RNA Generation, pure DNA samples[edit]

4. transposition reaction, using (T7tspn-top2) !!!USED 6 NG JURKAT DNA!!!!

  • add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
1.2 uL Nuclease free water
.8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL)
___________
5 uL total solution
method used in shendure paper:
4 uL nextera HMW buffer
X uL genomic DNA at prepared quantities
X uL Nuclease free water
______
17.5 uL total solution

add 2.5 uL of prepared transposomes


5. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)


7. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  bring to 20 uL with Nuclease free water
  X   uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  1   uL   10 mM ATP
  1   uL   10 mM CTP
  1   uL   10 mM GTP
  1   uL   10 mM UTP
  2   uL   T7 Enzyme Mix


b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)


  • Incubated for ~ 19 hours


  • Split RNA sample into 3 smaller samples of 6 uL each and used for each purification method.



Results[edit]

  • After IVT and before Zymo/AMPURE cleaning. Used 1 uL of sample to run TBU gel. Can combine samples for downstream processing.


  • quantification
  • does not seem right so will estimate about 2 ug per sample for testing.
' ng/ul in 20 uL
1. pure Jurkat DNA 901.0242134 18020.48427
2. pure Jurkat DNA 865.0337838 17300.67568


File:ZhangLab 2 2013-05-10 16hr 14min-labeled.jpg



  • Used only sample 1 of pure Jurkat DNA, IVT generated RNA for testing. Split the sample into 3 samples each, and used 6 uL for each purification method.


  • For gel: Used 2 uL of sample for samples 1-3, and 10 uL of sample for samples 4,5.
  • seems to have worked well this time. possibly due to using 3 minutes for drying time.
  • possibly the quanitations were right in the first gel, since getting a total of 9 ug of DNA.. could be becuase of TBU though? would have to quantitate with qubit to make sure.


' ' intnsity ng/ul ' '
5 2668.94008
2.5 2016.35006 in 10 uL
1. RNA specifc beds used 341.4345531 90490.4727 1707.172765
2. DNA specific beads used 335.8823687 89041.15265 1679.411844
3. Zymo purification 510.5786941 134643.184 2552.893471 34.22% sample loss when using beads
52.01% sample gain when using zymo



File:ZhangLab 2 2013-05-17 15hr 39min-labeled.jpg


conclusions[edit]

  • both bead methods work fine for isolating IVT generated RNA. Recovered the same amount ish. possibly reducing drying time to 3 minutes led to robust sample recovery.


  • Using beads results in ~35% sample loss compared to Zymo purification kit.


  • leftover transposon band (RNA or DNA) at ~80 bp is removed by using beads and thus will provide better sample for downstream RNA fragmentation. Band could also be RNA transposon at 80 bp since it is TBU gel, however regardless of being RNA or DNA it is removed.


  • have to be careful with using beads for low input samples since there is more sample loss than Zymo.


  • UPDATE 6/26/2013 assumed that it worked since the sample was diluted. split the sample into 3 aliquots of 6 uL each and assumed ddH2O was added to bring it to 20 uL, and 20 uL on beads was added for purification. Thus total dilution is 6.66X. For beads testing experiment When I added 30 uL of water, and 100 uL of beads to the sample during testing the total dilution is 7.5 which is comparable.