Brandon:LabNotes/Project1/2014-9-15

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


  • Think maybe transposase has already bound transposon? or contamination of nucleases.


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, transposase ONLY

  • using same protocol as above for transposition, protease. using transposase only
  • 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



Fragmentation testing for R4 f2 with 55C incubation, and with no Mg++ (only N-H2O). transposase only

  • using the same protocol as above except tagmentation for 5 minutes at 55C. And some samples are transposase and DNA only, no buffers
  • something in transposase solution is degrading DNA that does not require magnesium or any buffers.
1. R4 f2 1x dil., 6 ng DNA
2. R4 f2 2x dil., 6 ng DNA
3. R4 f2 4x dil., 6 ng DNA
4. R4 f2 8x dil., 6 ng DNA
5. R4 f2 16x dil., 6 ng DNA  
6. R4 f2 1x dil., 6 ng DNA, DNA + tsase only
7. R4 f2 2x dil., 6 ng DNA, DNA + tsase only
8. R4 f2 4x dil., 6 ng DNA, DNA + tsase only
9. R4 f2 8x dil., 6 ng DNA, DNA + tsase only
10. R4 f2 16x dil., 6 ng DNA, DNA + tsase only
11. 6 ng DNA ONLY control

File:ZhangLab 2 2014-09-16 19hr 43min-labeled.jpg




Fragmentation testing for R4 f2 95C 20m incubation, transposase only

  • trying heat denaturation of contamination. tsase dilutions incubated at 95C for 20 mins before transposition reactions. transpostion at 55C, the protease digestion.
  • whatever is degrading DNA is really strong.
1. R4 f2 1x dil., 6 ng DNA
2. R4 f2 2x dil., 6 ng DNA
3. R4 f2 4x dil., 6 ng DNA
4. R4 f2 8x dil., 6 ng DNA
5. R4 f2 16x dil., 6 ng DNA 
6. R4 f2 32x dil., 6 ng DNA
7. pure DNA only, 6 ng


File:ZhangLab 2 2014-09-17 12hr 57min-labeled.jpg



Fragmentation testing for R4 f2 95C 20m incubation, then protease digestion, transposase only

  • took above samples (already 95C for 20m, and did normal protease treatment on them. transpostion at 55C, the protease digestion.
  • finally degraded some of the contaminants. however at 1X concentration still there.
1. R4 f2 1x dil., 6 ng DNA
2. R4 f2 2x dil., 6 ng DNA
3. R4 f2 4x dil., 6 ng DNA
4. R4 f2 8x dil., 6 ng DNA
5. R4 f2 16x dil., 6 ng DNA 
6. R4 f2 32x dil., 6 ng DNA
7. pure DNA only, 6 ng


File:ZhangLab 2 2014-09-17 16hr 42min-labeled.jpg



Guanidine HCl purification after transposition on R2 f2, R4 f1, R4 f2 isolations of tsase[edit]

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?
  • R4 f1 works though 1X concentration doesn't, maybe still alot of nucleases?

File:ZhangLab 2 2014-09-15 15hr 31min-labeled.jpg File:ZhangLab 2 2014-09-17 13hr 00min-labeled.jpg



conclusions


  • actually looks like nuclease contamination based on several lines of evidence.

1. Nanodrop does not show any DNA bound to transposase. no peak at 260, only at 280.


2. Guanidine HCl purification is able to remove contamination and allow IVT RNA to be generated


3. Both DNA and RNA are degraded when only the transposase solution is added to samples. tn5 is DNA specific, and isn't known to degrade RNA.


4. Incubation at 95C for 20 minutes which should inactivate transposase didn't reduce fragmentation of DNA


5. Incubation of only genomic DNA and transposase solution degrades DNA. Magnesium is needed for activation of transposase, and is not added.


6. When fragmenting with transposase + transposon there is less fragmentation then when using transposase solution alone. This is probably because the nucleases are degrading genomic DNA in addition to added transposon thus more genomic DNA is leftover. With transposase solution only, the nucleases only have genomic DNA to degrade. As seen above and with fragmentation profiles of bound transposome complexes, with R2 f2, transposase + transposon stops degrading DNA at 2X dilution, and with transposase only it stops degrading DNA at 4X dilution. It is the same with R4 f2 its 16X versus 8X.



  • Thus need to figure out how to better purify transposase. In the mean time can use 4X or 8X dilution of R2 f2 for experiments, with the Guanidine HCl protocol with taq2x and IVT on beads.


Other thoughts


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



  • 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.