Jeff:LabNotes/Microbiome/2011-12-6

From ZhangLabWiki
Revision as of 23:50, 9 December 2011 by >Jgole
Jump to navigation Jump to search

In Situ Tagmentatation

  • 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

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
  • The amplicon seems to be broken up, as represented by the dots as opposed to a continuous fluorescence signal
  • 2 amplicons were extracted due to evaporation issues


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.
  • The gel looks funny. It does not seem that tagmentation worked due to the lack of a smear.

File:12-7-11 npcr.jpeg File:12-7-11 gel.jpeg

Microwell MDA with N6

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