Jeff:LabNotes/Microbiome/2013-2-19

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Revision as of 18:51, 19 February 2013 by >Jgole (Created page with "=='''Plan'''== *Perform MDA in microwells of NeuN - and fresh e coli samples using new epicenter kit *Second strand synthesis with PolI/ecoli ligase (low concentration) and n...")
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Plan[edit]

  • Perform MDA in microwells of NeuN - and fresh e coli samples using new epicenter kit
  • Second strand synthesis with PolI/ecoli ligase (low concentration) and new PCR
  • For tagmentation, use 1x buffer conc. and 5 min incubation since we need to mimic original protocol

Microwell MDA[edit]

  • Tested all of the reagents and everything (from MDA and tagmentation) is clean
  • This is the standard procedure that I will use for microwell MDA
  • All reagents are new
  • Pretreat with Oxygen Plasma. This is done in Nano3 with Roger's help
  • Use 8 arrays: 4 60831 NeuN-, 4 e coli

Put everything together.

  • Have successfully performed MDA cleanly in microwells with all sub parts amplifying cleanly.
  • Perform everything in hood, and all post MDA extractions in microscope hood
  • Cover PDMS with scotch tape after fabrication

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. The scotch tape should prevent this for the most part
  • Remove scotch tape from the arrays which will be used with a razor. Keep tape covering rest of arrays
  • UV 1% BSA (made from powder) and add 10 uL to each array in which cells will be seeded. Vacuum seed for 30s so that the BSA penetrates the wells. Incubate at rt in hood for 1 hr
  • Wash with 10 uL UV'd PBS at least 3x to remove excess BSA. Vacuum for 30 s
  • Remove PBS. Vacuum for several min until wells are dry. If you can still see dried BSA, repeat washing
  • 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. UV treat microscope stage area.
  • Dilute cells in PBS to 10 cell/uL.
  • Spread 3 uL cells evenly over array.
  • Let cells sit for 10 min so that they sink into the wells. At this point, most of the PBS should have evaporated
  • Freeze/thaw on dry ice 3x: 1 min on, 1 min off
  • Add 4.5 uL ALS on ice, wait 10 min, then 4.5 uL NS. Mix by stirring with pipette tip to ensure neutral pH before adding enzyme
  • Create the 1x master mix: 6 uL 200 uM N6 primer, .1 uL 50x Sybr, 1 uL dNTP, 2.3 uL buffer, 1.8 uL phi29.
  • Add ~50 uL mineral oil to completely cover square
  • 30 C for 10 hr, take fluorescent (GFP) image every 30 min, 200 ms exposure, 3 positions for each sample