Jeff:LabNotes/Microbiome/2011-11-7

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Microwell MDA with N6[edit]

Put everything together.

  • Have successfully performed MDA cleanly in microwells with all sub parts amplifying cleanly.
  • Grew fresh E coli and washed 2x in UV'd, filtered PBS
  • I found that the contamination is most likely coming from the post amplification steps, specifically unclean clean pipette tips during extraction and ethanol precipitation
  • These can be controlled: omit ethanol precip (found that this step is not necessary) and UV tips after a clean breaking procedure
  • Recently made new clean PDMS at half thickness. These should be easier to heat and much cleaner than the ones before.

Preparation

  • View microwells under microscope. Make sure there is limited debris in the wells. Also check under GFP filter to ensure there are no prior large DNA fragments
  • Apply ethanol to array. Let sit for 10 min
  • Apply vacuum to array for 30 s so the ethanol penetrates the wells.
  • UV clean water and microwell array for 10 min
  • Wash out the ethanol with clean water several times. Then add water and put on shaker for 10 min at low shaking speed. Transfer out of the hood in a clean (UV'd/ethanol/DNAway) pipette tip box
  • Vacuum for 30 s.
  • Remove the water by blowing with air from chemical hood.
  • Add .3 uL 30% BSA to 20 uL water. Add 10 uL of mix to well and UV for 5 min. Then let the BSA soak for 30 min.
  • We are now ready to move onto MDA

MDA

  • UV everything needed in reaction that does not contain DNA or enzymes (water, BSA, buffers, etc). Also UV/DNAway hood, pipettes, tweezers, microscope stage insert and cover, etc.
  • Obtain a flat dry ice brick (or put ice block in -80 C for 30 min). Place on a styrofoam lid and UV for 10 min. Also UV the metal thermocycler slide holder for 10 min and wipe with DNAway. UV slide stage adaptor as well.
  • Dilute cells in PBS to 20 cell/uL. Make the following mix. 22uL water, 6 uL diluted cells, and 2 uL BSA.
  • UV/ozone treat, transporting in clean pipette box. 5 min prime and 3 min treatment.
  • UV array for 5 min
  • Spread 5 uL cells evenly over array
  • Clean vacuum with EtOH and DNAway. Vacuum seed for 30 s.
  • Perform freeze/thaw lysis. Place array on dry ice with tweezers for 1 min. Remove and place on slide holder for 1 min. Repeat 2x.
  • Add 5 uL ALS, wait 3 min, then 5 uL NS.
  • Create the master mix: 5.8 uL water, 5 uL 200 uM N9 primer, .6 uL 50x Sybr, .8 uL dNTP, 3.4 uL buffer, 1 uL phi29.
  • Cover with clean mineral oil
  • 30 C for 10 hr, 65 C 20 min, 4 C forever