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

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==Procedure==
==Procedure==
*Prepare more E.coli gDNA MDA amplicons for testing.
*Prepare more E.coli gDNA MDA amplicons for testing.
 
**Preparing UV-ed ALS, NS buffer
 
 
50uL-MDANo-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.


===Operation manual===
===Operation manual===

Revision as of 16:24, 12 September 2010

MDA amplicons shearing using Misonic sonicator 3000 - test2

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-MDANo-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.

Operation manual

  • Hook up H20-tubes of cooling system. Release the white clipper.
  • Fill up H2O(tap H2O is acceptable) in the cup-horn to match the yellow tags
  • Turn on the switch of the cooling system.
  • Cooling system setting: Pump speed: "50", Desired temp "4C".
  • Turn on the switch of main controller.
  • Program setting:
    • Press "No" for not using micro-probe.
    • Press "program" to set up new conditions.
    • Tested condition: Pulse on(1min), Pulse(10sec), Output power(5), Temp monitoring(No). Total time(30min).
  • Press "start" to start the shearing.
  • Press "pause" to pause the shearing and aliqate sheared samples for validation.
  • After using, use the opposite order of operation procedures to turn off the machine.
    • Press "Stop" to stop shearing.
    • Turn off the main controller.
    • Turn down pump speed to "0" and turn off the switch.
    • For the cooling system, disconnect the H2O-tubes. Drain the H2O into the beaker.
    • Wipe out spilled H2O.
  • Technical support info: 800-323-4340 or 203-426-0101

Results

  • Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)
File:Sam091010-sheared DNA.jpg
  • It seems that even 7min (in the current pulse condition) is still too much for getting a 1~2Kb size fragments.
    • This result is reasonable because the Manual of Bioruptor sonicator(a very similar device)suggests to shear <5 min to get a 2~3Kb size (with 30sec on and off pulse).
  • I will try to optimize the shearing setting using new samples.