Brandon:LabNotes/Project1/2014-9-10

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testing 9/3/2014 (Round 4) isolated transposase with guanidine HCl purification, taq2x, IVT on beads[edit]


  • trying freshly isolated transposase to see if guanidine HCl denatures/removes RNases OR just simply allows IVT to proceed after tagmentation.




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
  • used with sc1-T7iBR-Idx25 before. will use t7top2 for testing now.
all with t7top2(8/22), t7-bot (8/22, NoP)
all follow protocol tagmentation, Guanidine HCl, pooling if applicable, beads pur., taq2x, IVT

samples
1. f1, 1 ng Jurkat DNA, t7top2, for pooling
2. f1, 1 ng Jurkat DNA, t7top2, for pooling
3. f1, 1 ng Jurkat DNA, t7top2, for pooling
4. f1, 1 ng Jurkat DNA, t7top2, for pooling
5. f1, 1 ng Jurkat DNA, t7top2, for pooling
6. f1, 1 ng Jurkat DNA, t7top2, for pooling
7. f1, 6 ng Jurkat DNA, t7top2
8. nextera 6 ng Jurkat DNA



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


Generation of custom barcode (3' side of fragments) illuminia adaptor transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (sc1-T7tspn-NoP-bot, "pMENTS") and (sc-ILA-adaptor).
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 50 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
1.25 uL of annealed Nextera transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • 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. Add Guanidine HCl. Add 5 uL guanidine HCl. pool samples if needed. samples that don't need to be pooled, bring to 15 uL with N-H20, then add 15 uL 8M guanidine HCl

    • If using beads next add 1.8X (54 uL) beads or for sample, or for pooled samples 96 uL beads.


ELUTE IN 9.6 or 12 uL DEPENDING, LEAVE BEADS IN REACTION


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase or 3.0 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]

  • TBU after IVT.
  • total RNA. Didn't really work. contamination? nextera worked fine as expected, and is extremely bright.
  • maybe the transposase is already bound to endogenous transposons in e coli? thus outcompeting the t7-top2 transposon?

File:ZhangLab 2 2014-09-11 19hr 19min-labeled.jpg