Sam:LabNotes/Microbiome-new/2010-9-10: Difference between revisions

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>Sam Chiang
>Sam Chiang
Line 10: Line 10:
**Preparing UV-ed ALS, NS buffer
**Preparing UV-ed ALS, NS buffer
   
   
  50uL-MDANo-SYBR recipe:
  50uL-MDA No-SYBR recipe:
                             1 rxn  4.5rxn         
                             1 rxn  4.5rxn         
  ---------------------------------------------
  ---------------------------------------------

Revision as of 00:03, 6 October 2010

MDA amplicons shearing using Misonic sonicator 3000 - with 30sec(on/off) setting

Background

  • The test on 09-09-'10 showed that amplicon was over-sheared using Palsson lab protocol (pulse on 1min, pulse off 10sec)
  • I noticed that this sonificator is very similar to Bioruptor I used before. After checking Bioruptor manual, I found that it needs only <5min shearing for getting 1~2Kbfragments.
  • An example of Bioruptor progarm for 300~400bp shearing.http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Human/2009-4-25

Procedure

  • Prepare more E.coli gDNA MDA amplicons for testing.
    • Preparing UV-ed ALS, NS buffer
50uL-MDA No-SYBR recipe:
                            1 rxn   4.5rxn         
---------------------------------------------
RT-PCR grade H2O           29.0    130.5        
10x RepliPhi phi-29 buffer  5.0     22.5
1mM  N6 primer              2.5     11.25
RepliPhi Phi-29 (100U/ul)   2.5     11.25    
dNTP (25mM)                 2.0      9.0 
----------------------------------------------
                           41.0    184.5/4.5=41 

3uL ALS buffer + 3uL E.coli gDNA (1ng/uL) + 3uL NS buffer 
Transfer 41uL master mix into lysed template.
Incubate at 30C for 8hr. Inactivate by 85C 3min.
  • Pool the four tubes of MDA amplicons into one and quantify the yield using QUBIT-HS dsDNA
    • Result: 666ng/uL in 200uL.
  • Take 1ug Amplicon and dilute into 100uL H2O in a 1.5mL-tube for shearing.

Amplicon shearing test

  • Direct shear the raw MDA amplicons without purification.
  • Following operation manual from 09/09/'10 with following changes on settings.
    • Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(10min).
    • Take out 20uL (200ng) sample for validation every 2min.

Results

  • Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)
File:Sam091010-E gel.jpg
  • The 2min shearing with 30sec (on/off) setting can get the 1~2Kb fragment size, which is suitable for Sanger sequcncing.