Sam:LabNotes/Microbiome-new/2010-6-9 exp2: Difference between revisions

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>Sam Chiang
(Created page with '='''50uL-MDA 10hr amplification on VA-sorted (EtOH fixed) E.coli (06-09-'10 sorted)'''= ==Background== *Repeat the single E.coli genome amplification experiments. The previous a…')
 
>Sam Chiang
 
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==Background==
==Background==
*Repeat the single E.coli genome amplification experiments. The previous amplification on 06-03-'10 wasn't successful.  
*Test the reagent cleanse by contamination level test.
*Test the MDA amplification on new sorted E.coli (EtOH-fixed) cells.


==Procedures==
==Procedures==
*Dilute Bacteria gDNA from 1ng/uL to 10pg/uL -> 100fg/uL -> 1 fguL -> 0.01 fg/uL
*Step1: Thaw reagents.
*Step1: Thaw reagents.
*Step2: Set up 1.5mL-tubes(x5), PCR-tube caps, and 96-well rack, ALS-NS UV-set (tray + strip tubes)
*Step2: Set up 1.5mL-tubes(x5), PCR-tube caps, and 96-well rack, ALS-NS UV-set (tray + strip tubes)
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  With SYBR recipe:
  With SYBR recipe:
                                                  
                                                  
                                     1 rxn         16+2 rxn         
                                     1 rxn x (8+5 +2)       
       -------------------------------------------------------------
       ----------------------------------
       RT-PCR grade H2O              26.5         477.0 uL         
       RT-PCR grade H2O              26.5            
       10x RepliPhi phi-29 buffer    5.0           90.0 uL             
       10x RepliPhi phi-29 buffer    5.0                    
       1mM  primer                    2.5          45.0 uL 
       1mM  primer                    2.5           
       2X SYBR Green I                2.5           45.0 uL                 
       2X SYBR Green I                2.5                        
       RepliPhi Phi-29 (100U/ul)      2.5          45.0 uL       
       RepliPhi Phi-29 (100U/ul)      2.5           
       --------------------------------------------------------------
       -----------------------------------
                                     39.0         702.0 uL  (702/18=39)         
                                     39.0                  
                                                                    
                                                                    
       25mM dNTP                      2.0           36.0 uL --- add dNTP later
       25mM dNTP                      2.0 --- add dNTP later


*Step8: Prepare the sample on rack at RT.
*Step8: Prepare the sample on rack at RT.
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*Setp12: Incubate at '''30C for 10hr'''. Followed by 85C 3min inactivation.
*Setp12: Incubate at '''30C for 10hr'''. Followed by 85C 3min inactivation.


==Results==
[[File:.sam060910-MDA contamination test.JPG|400px]] [[File:.sam060910-MDA 50uL MDA.JPG|400px]]


==Results==
[[File:sam060710-RLV-fixed Ecoli-50uL MDA strip1.jpg|400px]] [[File:sam060710-RLV-fixed Ecoli-50uL MDA strip2.jpg|400px]]


*There still has no sufficient separation between 1cell and NTC.
*The results showed 1~100fg contamination in both my sample and standard curves.
**I am wonder if it has something to do with fixation method. In the recent tests samples are all prepared using RLV-fixation.  
**The contamination may exist in the reagents I used.
**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.

Latest revision as of 23:31, 12 June 2010

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.