Sam:LabNotes/Microbiome-new/2010-6-9 exp2
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50uL-MDA 10hr amplification on VA-sorted (EtOH fixed) E.coli (06-09-'10 sorted)
Background
- Test the reagent cleanse by contamination level test.
- Test the MDA amplification on new sorted E.coli (EtOH-fixed) cells.
Procedures
- Step1: Thaw reagents.
- Step2: Set up 1.5mL-tubes(x5), PCR-tube caps, and 96-well rack, ALS-NS UV-set (tray + strip tubes)
- Step3: Making frest 5M KOH and ALS buffer
- 5M KOH: Weigh X gram KOH pellet. Add nuclease-free H2O: X/56.11 / 5 * 10^6 (KOH=56.11)
- ALS: Mix 200ul nuclease-free H2O, 25uL 1M DTT, 20ul 5M KOH, 5 ul 0.5M EDTA
- Step4: Test the pH of ALS + NS (expected to be ~7.5). UV-treatment on ALS and NS in 20uL scale (15min).
- Step5: UV-treated the PCR hood (15min)
- Step6: Pool the UV-ed ALS and NS.
- Step7: Prepare incomplete master mix - 50uL-reaction vol.
With SYBR recipe: 1 rxn 16+2 rxn ------------------------------------------------------------- RT-PCR grade H2O 26.5 477.0 uL 10x RepliPhi phi-29 buffer 5.0 90.0 uL 1mM primer 2.5 45.0 uL 2X SYBR Green I 2.5 45.0 uL RepliPhi Phi-29 (100U/ul) 2.5 45.0 uL -------------------------------------------------------------- 39.0 702.0 uL (702/18=39) 25mM dNTP 2.0 36.0 uL --- add dNTP later
- Step8: Prepare the sample on rack at RT.
- Step9: Set up timer for 5min
- Step10: 3uL ALS lysis for 5min. Followed by 3uL NS nutralization.
- Step11: Replace the tubes onto cold block.
- Step12: Add dNTP into master mix
- Step13: Transfer 41uL complete master mix into each reaction.
- Step14: Cap the tube caps and seal tightly.
- Setp12: Incubate at 30C for 10hr. Followed by 85C 3min inactivation.
Results
File:Sam060710-RLV-fixed Ecoli-50uL MDA strip1.jpg File:Sam060710-RLV-fixed Ecoli-50uL MDA strip2.jpg
- There still has no sufficient separation between 1cell and NTC.
- I am wonder if it has something to do with fixation method. In the recent tests samples are all prepared using RLV-fixation.
- The other possibility is that the plate has been contaminated, or the MDA processing is not clean.
- I am thinking to do a contamination test to make sure my reagents are clean.