Brandon:LabNotes/Project1/2014-6-18

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Dialysis of last fractions, and Tn5 activity testing[edit]

  • Dialysis was performed on the last tn5 fractions from the chromatography column. This includes the fractions obtained from letting the column sit overnight in 3-4 mL cleavage buffer after collection of the first fractions.
    • 8.44 uM was isolated from the last fractions
    • 7.186 uM was isolated from the column left overnight



  • will test the fractions for enzyme activity as it is hoped that there will still be tn5 left over, otherwise it will have to be isolated again.


Before starting protocols[edit]

1. Check if have enough reagents etc for the protocol


  • 2. Samples
  • transpoase was either directly annealed to transposon, diluted in storage buffer then annealed to transposon, diluted 2X more after being diluted in storage buffer, or 4X more after being diluted in storage buffer.
samples (all use buffer D6)
1. T7tspn-top2, E1 direct anneal (4.22 uM), 6 ng DNA
2. T7tspn-top2, E1 strg-anneal (1.77 uM), 6 ng DNA
3. T7tspn-top2, E1 2X dil. strg-anneal (.88 uM), 6 ng DNA
4. T7tspn-top2, E1 4X dil. strg-anneal (.44 uM), 6 ng DNA
5. T7tspn-top2, E2 direct anneal (3.6 uM), 6 ng DNA
6. T7tspn-top2, nxta direct anneal, 6 ng DNA
7. T7tspn-top2, nxta strg-anneal, 6 ng DNA
8. T7tspn-top2, nxta 2X dil. strg-anneal, 6 ng DNA
9. T7tspn-top2, nxta 4X dil. strg-anneal, 6 ng DNA
10. pure DNA only, 6 ng DNA
11. NTC



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 30 minutes at 37C (DID THIS TIME)
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


4. transposition reaction Add all components and incubate at 55C for 6 minutes

1.0 uL 5X Custom Tagmentation buffer
2.0 uL lysed cells/pure genomic DNA
1.0 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted)
1.0 uL Nuclease free water
___________
5.0 uL total solution


5. Protease digestion of proteins/transposase

To each tube, add:
1 uL  Proteinase K
Incubate: 50C 10 minutes, Did not do 70C inactivation of protease



Results[edit]

  • TBE gel
  • did not work.

File:ZhangLab 2 2014-06-18 19hr 30min-labeled.jpg


conclusions[edit]

  • isolatedtn5 did not work while nextera tn5 did. Thus will have to reisolate


  • other possibility is T7tspn-top2 could be getting old and not working, thus will try reannealing to nextera tn5 as a control and testing again with purified tn5.