Sam:LabNotes/Microbiome-new/2010-11-17: Difference between revisions

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>Sam Chiang
>Sam Chiang
 
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*I didn't do any additional amplification on the short-amplified E.coli SAGs since the over-dose MDA reaction buffer may interfere the library construction.  
*I didn't do any additional amplification on the short-amplified E.coli SAGs since the over-dose MDA reaction buffer may interfere the library construction.  


==QUBIT Sample quantification==
==Procedures==
  [[File:sam111710-qubit.jpg|400px]]
===QUBIT quantification===
  [[File:sam111710-qubit-new.jpg|900px]]
*IC = internal control of E.coli gDNA


*4 pairs of samples(marked in red) were prepared for shearing:
**WGA102710-(2-1)- 3hr and 10hr
**WGA102710-(2-4)- 3hr and 10hr
**WGA110310-(2)- 4hr and 10hr
**WGA110310-(4)- 4hr and 10hr


===Procedures===
===Sample preparation===
*Label the Covaris microtubes. Open the caps and leave tubes on 96-well rack.  
*Label the Covaris microtubes. Open the caps and leave tubes on 96-well rack.  
*UV-treat the tubes and pipettes for 15min.
*UV-treat the tubes and pipettes for 15min.
**Thaw the tRNA carrier (stored in -80C). Use the "2"-tRNA(96ng/uL)
**Thaw the tRNA carrier (stored in -80C). Use the "2"-tRNA(96ng/uL)


   Sample no.  Sample name          Conc.(ng/uL)  Sample(uL)   330ng-tRNA("2")  H2O    Total DNA(ng)
   Sample no.  Sample name          Conc.(ng/uL)  Sample(uL)     tRNA("2")  H2O    Total DNA(ng)
   ---------------------------------------------------------------------------------------------------
   ---------------------------------------------------------------------------------------------------
   HC          sam080610_2-1_2h     0.685         100          3.43             0     68.5ng
   HC01        WGA102710-(2-1)- 3hr     0.88         50.00          3.5      0.00    43.95ng
   2-S          sam080610_2-2_2h     0.703         100          3.43              0     70.3ng
  HC02        WGA102710-(2-1)-10hr  729.07          0.68           -      49.00  500.00ng
   3-S         sam081210_1-5_2h     0.704         100         3.43              0     70.4ng
   HC03        WGA102710-(2-4)- 3hr     0.98         50.00          3.5      0.00    48.95ng
   4-S         sam081210_2-5_2h    0.693        100          3.43              0      69.3ng
   HC04        WGA102710-(2-4)-10hr  693.02         0.72            -      49.00  500.00ng
  HC05        WGA110310- (2) - 4hr     2.16        50.00            -        0.00  108.14ng
  HC06         WGA110310- (2) -10hr  661.63         0.75            -      49.00  500.00ng
  HC07        WGA110310- (4) - 4hr    3.09        50.00            -        0.00  154.65ng
   HC08        WGA110310- (4) -10hr  974.42         0.52            -      49.00  500.00ng
    
    
   1-L          sam080610_2-1_10h  220.0          2.27          -              97.7  500.0ng
   *I didn't put any tRNA carrier for 4hr-amplified amplicons.
  2-L          sam080610_2-2_10h  322.0          1.55          -             98.5  500.0ng
*'''50uL''' sample suspension were carefully transferred into microtubes.
  3-L          sam081210_1-5_10h  540.0          0.92          -              99.0  500.0ng
  4-L          sam081210_2-5_10h  551.0          0.90          -              99.0  500.0ng
*'''100uL''' sample suspension were carefully transferred into microtubes.
*Samples tubes were tapped on the rack and packed in a paper box with three disposable cooler.
*Samples tubes were tapped on the rack and packed in a paper box with three disposable cooler.
*The package was shipped to the Sara through FedEx(track# 4490-5094-2339 on 10-19-'10).
*The package was shipped to the Sara through FedEx 11-17-'10 by Alice.
 
==Follow up(11-18-'10)==
*I noticed that I accidently did the wrong dilution for all of the 10hr-amplification samples. I load much less thant 500ng for shearing.
  HC02        WGA102710-(2-1)-10hr
  HC04        WGA102710-(2-4)-10hr
  HC06        WGA110310- (2) -10hr 
  HC08        WGA110310- (4) -10hr 
*Since the concentrations are very high for these samples, I am planning to do the local Bioruptor shearing for these samples later.
 
==Follow up(12-07-'10)==
*Since Alice is going to shear her samples today. I prepared those four missed samples again in microtubes and sent to Sarah for shearing.
                                      H2O  DNA for 500ng
  HC02        WGA102710-(2-1)-10hr  49.0  0.68 uL
  HC04        WGA102710-(2-4)-10hr  49.0  0.72 uL
  HC06        WGA110310- (2) -10hr  49.0  0.75 uL
  HC08        WGA110310- (4) -10hr  49.0  0.52 uL
 
*Total volume 50uL. Shipped in RT (since all of them are in high DNA amount).

Latest revision as of 22:53, 7 December 2010

MDA samples preparation for Covaris shearing-(single E.coli amplicons)[edit]

Background[edit]

Procedures[edit]

QUBIT quantification[edit]

File:Sam111710-qubit-new.jpg
*IC = internal control of E.coli gDNA
  • 4 pairs of samples(marked in red) were prepared for shearing:
    • WGA102710-(2-1)- 3hr and 10hr
    • WGA102710-(2-4)- 3hr and 10hr
    • WGA110310-(2)- 4hr and 10hr
    • WGA110310-(4)- 4hr and 10hr

Sample preparation[edit]

  • Label the Covaris microtubes. Open the caps and leave tubes on 96-well rack.
  • UV-treat the tubes and pipettes for 15min.
    • Thaw the tRNA carrier (stored in -80C). Use the "2"-tRNA(96ng/uL)
 Sample no.   Sample name          Conc.(ng/uL)  Sample(uL)      tRNA("2")   H2O    Total DNA(ng)
 ---------------------------------------------------------------------------------------------------
 HC01         WGA102710-(2-1)- 3hr     0.88         50.00           3.5       0.00    43.95ng
 HC02         WGA102710-(2-1)-10hr   729.07          0.68            -       49.00   500.00ng
 HC03         WGA102710-(2-4)- 3hr     0.98         50.00           3.5       0.00    48.95ng
 HC04         WGA102710-(2-4)-10hr   693.02          0.72            -       49.00   500.00ng
 HC05         WGA110310- (2) - 4hr     2.16         50.00            -        0.00   108.14ng
 HC06         WGA110310- (2) -10hr   661.63          0.75            -       49.00   500.00ng
 HC07         WGA110310- (4) - 4hr     3.09         50.00            -        0.00   154.65ng
 HC08         WGA110310- (4) -10hr   974.42          0.52            -       49.00   500.00ng
 
 *I didn't put any tRNA carrier for 4hr-amplified amplicons.
  • 50uL sample suspension were carefully transferred into microtubes.
  • Samples tubes were tapped on the rack and packed in a paper box with three disposable cooler.
  • The package was shipped to the Sara through FedEx 11-17-'10 by Alice.

Follow up(11-18-'10)[edit]

  • I noticed that I accidently did the wrong dilution for all of the 10hr-amplification samples. I load much less thant 500ng for shearing.
 HC02         WGA102710-(2-1)-10hr 
 HC04         WGA102710-(2-4)-10hr 
 HC06         WGA110310- (2) -10hr  
 HC08         WGA110310- (4) -10hr   
  • Since the concentrations are very high for these samples, I am planning to do the local Bioruptor shearing for these samples later.

Follow up(12-07-'10)[edit]

  • Since Alice is going to shear her samples today. I prepared those four missed samples again in microtubes and sent to Sarah for shearing.
                                      H2O   DNA for 500ng
 HC02         WGA102710-(2-1)-10hr   49.0   0.68 uL
 HC04         WGA102710-(2-4)-10hr   49.0   0.72 uL
 HC06         WGA110310- (2) -10hr   49.0   0.75 uL
 HC08         WGA110310- (4) -10hr   49.0   0.52 uL
  • Total volume 50uL. Shipped in RT (since all of them are in high DNA amount).