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)[edit]

Background[edit]

  • Test the reagent cleanse by contamination level test.
  • Test the MDA amplification on new sorted E.coli (EtOH-fixed) cells.

Procedures[edit]

  • Dilute Bacteria gDNA from 1ng/uL to 10pg/uL -> 100fg/uL -> 1 fguL -> 0.01 fg/uL
  • 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 x (8+5 +2)        
      ----------------------------------
     RT-PCR grade H2O              26.5              
     10x RepliPhi phi-29 buffer     5.0                      
     1mM  primer                    2.5           
     2X SYBR Green I                2.5                          
     RepliPhi Phi-29 (100U/ul)      2.5           
     -----------------------------------
                                   39.0                   
                                                                 
     25mM dNTP                      2.0  --- 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[edit]

File:.sam060910-MDA contamination test.JPG File:.sam060910-MDA 50uL MDA.JPG


  • The results showed 1~100fg contamination in both my sample and standard curves.
    • The contamination may exist in the reagents I used.