Brandon:LabNotes/Project1/2015-3-16

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guanidine HCl treatment with EtOH purification, with taq2X, IVT after Round 2[edit]

  • Erin's samples didn't work, probably because IVT was done twice on them but who knows. will try again.


  • in experiments in march/april 2014 did not do guanidine HCl treatment, then purificaiton. did straight purification which lead to lots of product loss. Thus still try guanidine treatment with EtOH purification, then IVT.


  • also need to test this since this is how combination of C1 samples would occur.



Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • sc1 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples used for assay
all with t7top2 from Illuminia at 5X concentration.
all follow protocol tagmentation, Guanidine HCl, pooling if applicable, EtOH purf., taq2x, IVT

samples
1. 100 cells, tn5-059 t7-top2 3.5 uM rep1
2. 100 cells, tn5-059 t7-top2 3.5 uM rep2
3. 100 cells, tn5-059 t7-top2 3.5 uM rep3
4. 100 cells, tn5-059 t7-top2 3.5 uM rep4
5. 100 cells, tn5-059 t7-top2 3.5 uM rep5
6. 500 cells, tn5-059 t7-top2 3.5 uM with tRNA carrier
7. 500 cells, tn5-059 t7-top2 3.5 uM no carrier added carrier by accident
8. 1000 cells, tn5-059 t7-top2 3.5 uM
9. 6 ng pure DNA, tn5-059 t7-top2 3.5 uM
10. NTC, tn5-059 t7-top2 3.5 uM



IVT Protocol[edit]

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of sc1-T7iBR-IdxXX transposomes

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot.
    • b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1. Add 0.5 uL of 100% sterile glycerol to tube
2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
4. Add 1.0 uL of DILUTED Ez-TN5 transposase to well.
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

  • 1. count cells, spin down all cells at 250-500 g for 5 minutes
  • 2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XX cells/uL
  • 3. Count cells again, aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
  • 3. add LB to cell aliquots, mix briefly (mineral oil optional)
  • 4. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.


4. transposition reaction Add all components and incubate at 37C FOR 30 MINUTES

1.0 uL 2X Lysis buffer
1.0 uL genomic DNA/cells
1.0 uL 5X Custom Tagmentation buffer
1.0 uL prepared T7 transposomes (MAKE SURE TO ADD LAST)
1.0 uL N-H20
___________
5.0 uL total solution


5. TO ALL SAMPLES: Add Guanidine HCl. bring to 15 uL with N-H20, then add 15 uL 8M guanidine HCl.

  • combine samples 1-5.


5.5. Add 500 ng tRNA carrier to each sample. Then do standard EtOH purification. Elute into 6 uL N-H2O.

  • samples: 1-5 combined, 6,7,8. add carrier after combining samples.
  • before EtOH purification, bring to 450 uL with 1X TE.


6. Fill in reaction

  • Add 6.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera)


8. 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 10 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)



9. Clean with Zymo clean and concentrator

  • elute samples in 10 uL of N-H2O
  • quanitate with Qubit or on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


Results[edit]

  • TBE gel after IVT. used TBE since no TBUs were left.

File:ZhangLab 2 2015-03-17 17hr 22min-labeled.jpg


future[edit]

  • didn't work to well for any of the EtOH purified samples. maybe try using protease before hand then guanidine HCl for extraction. could be when guanidine HCl is diluted proteins can rebind to DNA.