Brandon:LabNotes/Project1/2014-11-14

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Fragmentation profile of epicentre ez-tn5 with dilutions[edit]

  • will have comparison to R6f1 transposome to see how it looks. with 6 ng pure DNA.


  • followup to Determining the concentration of ez-tn5 from epicentre, where it was determined that ez-tn5 is based on nanodrop and bradford is ~16X dilution of the self made tn5, and has an approximate concentration of ~0.5 uM of tn5. hopefully this analysis will confirm those findings.


  • ez-tn5 has not been performing well either, with IVT smears being less bright then those from the ez-tn5. not sure why. previous assays from a year ago are brighter or just as bright from the IVT smears, as the ones currently being generated with self made tn5.


1. dilute transposase to respective dilutions, 1X, 2X, 4X, 8X, 16X, 32X


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 (10 uM)
0.5 uL of 100% sterile glycerol
1.0 uL of Ez-TN5 transposase


3. make following reaction

  • reaction composition
1 uL pure Jurkat DNA (6 ng)
1 uL 2X Lysis buffer
1 uL 5X custom tagmentation buffer (buffer D6)
1 uL H2O
1 uL transposase
__________
5 uL total

incubated reaction at 55C for 5 mins, followed by addition of 1 uL qiagen protease digestion,
50C for 10 mins, 70C for 20 mins.
samples.
1. ez-tn5, undil tsase, 6 ng DNA
2. ez-tn5, 2X dil. tsase, 6 ng DNA
3. ez-tn5, 4X dil. tsase, 6 ng DNA
4. ez-tn5, 8X dil. tsase, 6 ng DNA
5. ez-tn5, 16X dil tsase, 6 ng DNA
6. ez-tn5, 32X dil. tsase, 6 ng DNA
7. 6 ng pure DNA alone


4. run on TBE gel

  • didn't work first time, repeating. though there is some light fragmentation in first 2 lanes. not sure why.

File:ZhangLab 2 2014-11-14 14hr 01min-labeled.jpg



  • second run, have more fragmentation but not complete fragmentation. maybe this batch of nextera isn't that good or its less concentrated? also haven't been seeing good smears after IVT from ez-tn5 when it has been used as the positive control.

File:ZhangLab 2 2014-11-14 16hr 35min-labeled.jpg File:ZhangLab 2 2014-11-14 16hr 37min-invert-labeled.jpg


  • when compared to the profile of R6f1 isolated tn5 we can see that ~16-20X dilution is equal the the current stock of nextera Tn5. maybe in the past it would have been equal to 8-12X dilution since ez-tn5 was able to completely fragment 6 ng of genomic DNA.
  • assay/gel was done by erin the undergrad, thus lanes 4 and 5 could be switched or mispipetted.
  • also gel was left overnight at 4C in TBE in plastic bag since we ran out of sybr gold

File:ZhangLab 2 2014-11-14 10hr 55min-labeled.jpg File:ZhangLab 2 2014-11-14 10hr 56min-invert-labeled.jpg


conclusions[edit]

  • nextera ez-tn5 is not performing to well. based on concentrations analysis and this fragmentation analysis, it is safe to say nextera ez-tn5 is ~16X dilution of R6f1, the current self made tn5 that is being used.