Jeff:LabNotes/Microbiome/2012-11-14

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Tagmentation test using Genomic DNA[edit]

  • Test 4 samples, each with 25 ng genomic DNA starting template
  • Sample 1: PC in water
  • Sample 2: DNA is EtOH precipitated and eluted in water
  • Sample 3: DNA in New prep below w/o Pol I. ALS and NS premixed as to be neutral
  • Sample 4: Pol I treated DNA using New prep

Pol 1 treatment

  • Start with 1 uL DNA
  • Add 1.5 uL ALS, 3min
  • Put on ice and add 1.5 uL NS

New Prep

Template  4 uL    
10x buffer 4 .7 uL    
dNTP .35 uL          
N6(unmodified) .35 uL  
water .4 uL         
Pol I .5 uL         
----------------------------------
Total 7 uL         


  • 37 C for 30 min, 65C for 10 min

Tagmentation

DNA in water  7 uL
HMW buffer  2 uL, (.6 uL for New prep samples for a total of 2 uL)
water (for new prep only) 1.4 uL
1:50 diluted enzyme  1 uL
----------------------
Total 10 uL
  • 55C for 5 min

Protease treatment

  • Add 1 uL 1:100 diluted protease
  • 10 min 50C, 20 min 70C

Klenow

  • Add 1 uL exo- klenow and 1 uL dNTP to each
  • 37C for 15 min, 75 for 20 min

1st PCR round

DNA  13 uL
KAPA Robust mix  15 uL
Orange  .6 uL
blue   .6 uL
50x SYBR .24 uL
--------------------------
Total  30 uL
  • 95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8

Second round PCR

  • Pause PCR program and add following to each
1st round PCR mix 30 uL
KAPA Robust mix 35 uL
Adapter 1 2 uL
Adapter 2 Barcode 2 uL
SYBR Green 1 .56 uL
water 30.44 uL
----------------------
Total 100 uL

1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min



Bead purfication

  • Add 100 uL beads to each, mix, and let sit at RT for 8min
  • Transfer tubes to magnetic rack and let sit for 5 min
  • Remove supernatant
  • 2x 80% EtOH wash
  • Sit for 15 min
  • Elute in 23 uL Qiagen buffer


Results

  • There is a 1 cycle difference between PC and EtOH, implicating 1/2 the DNA is lost
  • Even though the buffer for the Pol I reactions is the same as tagmentation buffer, tagmentation is very inefficient. ALS is prepared as diluted KOH only (no DTT or EDTA). Not sure what is happening. Only differences are K and CL ions from ALS/NS, and dNTP's/N6 primer from Pol I mix
  • When the Pol I reaction was run, it is entirely inhibited meaning the enzymes are somehow competing (even though Pol I is killed)
  • In any case, EtOH continues to be the best option, and I will only use this in the future


File:11-14-12 pcr.jpg File:11-14-12 gel1.jpg


Microwell MDA[edit]

Found that the skin microbiome samples is ~10x less concentrated that what is expected, most likely due to cell lysis. I will use different concentrations today

  • I have analyzed new data. It seems that the issue is with adapter annealing during PCR after tagmentation. I will create new amplicons and see if new PCR program is more efficient
  • During library construction, I will implement new techniques to try and increase the complexity of the library
  • 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
  • I will process these samples with HMW buffer during tagmentation
  • All reagents are new
  • Pretreat with Oxygen Plasma. This is done in Nano3 with Roger's help
  • I have skin bacteria and healthy neuronal/non neuronal libraries ready to be sequenced. Today will focus of alzheimer's samples
  • Use 8 arrays: 2 e coli, 2 1:5 diluted skin mb, 2 1:30 skin mb, 2 60831 NeuN- healthy neurons

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: 5 uL 200 uM N6 primer, .08 uL 50x Sybr, .8 uL dNTP, 2.2 uL buffer, 1.6 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