Jeff:LabNotes/Microbiome/2012-8-16

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Library Construction: Microwell Amplicons [edit]

  • MDA seems to have worked based on fluorescence.
  • Extracted 2 of each sample in 1 uL water
  • Nuclei had by far the greatest fluorescence in rtMDA
  • HMW buffer in tagmentation
  • Use Pol1 +ALS/NS treatment

Pol 1 treatment

  • Start with 1 uL DNA
  • Add 1.5 uL ALS, 3min
  • Put on ice and add 1.5 uL NS
Template  4 uL    
10x buffer 2 uL    
dNTP 1 uL          
N6(unmodified) 5 uL  
water 7 uL         
Pol 1 1 uL         
----------------------------------
Total 20 uL         


  • 37 C for 1 hr, 65C for 10 min
  • Standard EtOH (Add 200 ng tRNA carrier) and elute in 7 uL water. The tRNA was newly treated with DNAse

Tagmentation

DNA in water  7 uL
HMW buffer  2 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

1st PCR round

DNA  13 uL
KAPA Robust mix  25 uL
Primer cocktail  1 uL
1 uM barcode (25-37)  1 uL
water  9.6 uL
50x SYBR .4 uL
--------------------------
Total  50 uL
  • 95 C 30s (95 C 10s, 62C 15s, 72 C 2 min)x20


Bead purfication

  • Add 50 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

  • Tagmentation worked and got good results
  • Some amplified better than others
  • Save libraries for validation

File:8-16-12 pcr.jpeg File:8-16-12 gel 2.jpeg File:8-16-12 gel.jpeg