Brandon:LabNotes/Project1/2014-9-15

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how come guanidine HCl purification still doens't allow IVT to work?


  • Think maybe transposase has already bound transposon?


Protocol

4. transposition reaction add the below into one tube and incubate at 37C FOR 30 MINUTES.

1.0 uL washed cells
1.0 uL 2X Lysis Buffer
1.0 uL 5X custom tagmentation buffer
1.0 or 2.0 uL prepared transposomes
1.0 uL Nuclease free water
___________
5.0 uL total solution

5. Protease digestion of transposase, protease inactivation To each tube, add:

1 uL  Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O)
Incubate: 50C 10 minutes, 70C 20 minutes


6. Run on TBE gel to visualize fragmentation


  • does the transposase alone (with reaction buffer), fragment DNA?
  • it does, which it shouldn't.

File:ZhangLab 2 2014-09-12 15hr 06min-labeled.jpg



  • checking with nextera, and dilutions of R4 (9/3/2014) transposase to 16X. same protocol as above used.
  • nextera ez-tn5 doesn't fragment DNA. more pure? completely unbound transposase?

File:ZhangLab 2 2014-09-12 20hr 09min-labeled.jpg


Fragmentation testing again for R4 f2, and for R2 f2

  • using same protocol as above for transposition, protease
  • worked really well for R2 f2, which is the only isolation that has been working for IVT. other is R2 f1. must be either already bound transposon, or contamination or both.


samples
1. R2 f2 1x dil., 6 ng DNA
2. R2 f2 2x dil., 6 ng DNA
3. R2 f2 4x dil., 6 ng DNA
4. R2 f2 8x dil., 6 ng DNA
5. R2 f2 16x dil., 6 ng DNA 
6. R2 f2 32x dil., 6 ng DNA 
7. R2 f2 64x dil., 6 ng DNA 
8. R2 f2 128x dil., 6 ng DNA 
9. R4 f2 1x dil., 6 ng DNA
10. R4 f2 2x dil., 6 ng DNA
11. R4 f2 4x dil., 6 ng DNA
12. R4 f2 8x dil., 6 ng DNA
13. R4 f2 16x dil., 6 ng DNA 
14. R4 f2 32x dil., 6 ng DNA 
15. R4 f2 64x dil., 6 ng DNA 
16. R4 f2 128x dil., 6 ng DNA 
17. 6 ng DNA, with rxn buffer
18. 50/50 TE/glycerol (4 uL), + 6 ng DNA
19. 1 uL D6, 3 uL H2O, + 6 ng DNA
20. 6 ng DNA, with rxn buffer #2


File:ZhangLab 2 2014-09-15 15hr 50min-labeled.jpg File:ZhangLab 2 2014-09-15 15hr 55min-labeled.jpg




Guanidine HCl purification after transposition on R2 f2, R4 f2 isolations of tsase

IVT Protocol

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

Generation of t7tspn-top2 transposome

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of t7tspn-top2(8/22) and T7tspn-NoP-bot(8/22).
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 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
0.5 uL of annealed transposon
0.5 uL of 100% sterile glycerol
1.0 uL of Ez-TN5 transposase
  • 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

1. R2 f2 1x dil., 6 ng DNA
2. R2 f2 2x dil., 6 ng DNA
3. R2 f2 4x dil., 6 ng DNA
4. R2 f2 8x dil., 6 ng DNA
5. R2 f2 16x dil., 6 ng DNA
6. R4 f2 1x dil., 6 ng DNA
7. R4 f2 2x dil., 6 ng DNA
8. R4 f2 4x dil., 6 ng DNA
9. R4 f2 8x dil., 6 ng DNA
10.R4 f2 16x dil., 6 ng DNA
11. nextera positive control, 6 ng DNA


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 XXXX cells/uL
  • 3. Aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute LB to 2X, or as needed (from 10X stock)
  • 2. refrigerate or warm LB or keep at RT
  • 3. add lysis buffer 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 the below into one tube and incubate at 37C FOR 30 mins.
1.0 uL washed cells
1.0 uL 2X Lysis Buffer
1.0 uL 5X custom tagmentation buffer (buffer D6)
1.0 uL prepared transposomes
1.0 uL Nuclease free water
___________
5.0 uL total solution


5. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, use 1 uL of 11.1 ug/uL (.5 AU/ml) protease (dilute 5.5 uL 20 ug/ul with 4.5 uL N-H2O)
Incubate: 50C 10 minutes, 70C 20 minutes


6. Fill in reaction

  • Add 1 uL 5X taq polymerase, run at 72C for 3 minutes. (OR 6 uL taq2X) (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)



8. Clean with Zymo clean and concentrator now

  • 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


  • looks like it worked for R2 f2, and made R4 f2 work too though only when diluted.. why?

File:ZhangLab 2 2014-09-15 15hr 31min-labeled.jpg



conclusions


  • looks like replacing HEPES with tris for hte transposase isolation was a bad idea. maybe tris hcl allows transposase to bind endogenous transposon sequences in e coli?


  • A600 of 1.0 = 1 billion e coli per ml. thus 1 trillion in 1 liter.


  • sonication takes 20-30 minutes. pelleting for 30 minutes (~45 with traveling etc). then PEI precip. so transposase has ~1 hour 15 mins to bind transposon sequences.