Jeff:LabNotes/Microbiome/2011-12-8

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In Situ Tagmentatation[edit]

  • I believe that doing in situ tagmentation will help with contamination problem
  • The amplicons will be tagged prior to extraction, and if all the transposases are used up, there is no room for contamination in this step
  • I also want to see if protease helps

Tagmentation

DNA in water  6.5 uL
HMW buffer  2 uL
1:10 diluted enzyme  2 uL
50x SYBR green .5 uL
----------------------
Total 10 uL
  • Pipette 10 uL mix onto array
  • 55C for 5 min
  • Look under microscope
  • For protease, the library was added to 5 uL water, 1 uL 1:500 protease and subjected to standard protease temps
  • 2 amplicons were extracted due to evaporation issues, and 1 for protease, and an NTC


1st PCR round

DNA  6 uL
BST  1 uL
KAPA fast mix  15 uL
10 uM orange primer  .24 uL
10 uM blue primer  .24 uL
water  7.52 uL
--------------------------
Total  30 uL
  • 60 C 20 min, 72 C 3 min, 95 C 30s (95 C 10s, 58 C 30s, 72 C 2 min)x20, 72C 3 min

Results

  • The amplicons shot up meaning that tagmentation worked.
  • Protease seemed to inhibit PCR
  • The gel looks better, but still not right. There is a smear, but it starts about 100 bp too low. This is most likely because it took some time to remove the amplicons and the transposases were still active.
  • There is still a band need to figure this out

File:12-8-11 npcr.jpeg File:12-8-11 gel.jpeg

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 1% BSA to well and UV for 5 min. Then let the BSA soak for 1 hr.
  • 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 2 hr, 65 C 20 min, 4 C forever