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