Sam:LabNotes/Human/2009-5-22: Difference between revisions

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>Sam Chiang
(New page: ='''2nd MDA amplicons purification using MICROCON kit (#YM-100, Millipore)'''= ==Objective== *In order to estimate the DNA concentration of 2nd MDA amplicons. 20uL of 2nd MDA amplicon we...)
 
>Sam Chiang
Line 16: Line 16:
*Assemble the filter
*Assemble the filter


*Transfer all DNA (about 20 uL)into a 0.5 mL tube
*Transfer 20uL of MDA amplcion into a 1.5-mL tube.


*Add water up to 200 uL of total volume
*Add Nuclease-free H2O (Ambion) up to 200uL of total volume. Mix by vortexing for 10 sec and briefly spining down the mixture.


*Mix by p200 pipettor and transfer the mixture on the filter reservoir.  
*Transfer the mixture on the filter reservoir.  


*Cap the tube and centrifuge at 14,000xg for 12 min.
*Cap the tube and centrifuge at 14,000xg for 12 min.


*Add 20uL H2O on the filter. Invert and transfer the filter on a fresh collection tube. Centrifuge at 1000 xg for 3 min.
*Add 20uL Nuclease-free H2O (Ambion) on the filter. Stay the filter for one min in RT. Invert and transfer the filter on a fresh collection tube. Centrifuge at 3000xg for 3 min.


===set up PCR reaction===
*Estimate the volume of elutant using tips. Nanodrop the dsDNA concentration.


*Enzyme: Qiagen Multiplex PCR kit


*Primer: Primer mix "Y"
==Results==
 
*Template
**Row A: Un-purified: A1, D7(1/15), D7(1/30), B10(1/15), pos (29 ng gDNA), H2O
**Row B:    purified: A1, D7(1/15), D7(1/30), B10(1/15), pos (29 ng gDNA)
 
  Reaction volume test:
            10uL x 13
            ---------
  H2O        2    26
  Q-sol      1    13
  Enzyme    5    65


==Results==
==Results==

Revision as of 08:19, 26 May 2009

2nd MDA amplicons purification using MICROCON kit (#YM-100, Millipore)

Objective

  • In order to estimate the DNA concentration of 2nd MDA amplicons. 20uL of 2nd MDA amplicon were purified using MICROCON kit and then measured dsDNA concentration using Nanodrop.
  • It is suggesed to purify the MDA amplicon before nanodrop measuring. Otherwise the primer dimmer and salts in MDA amplicon might interfere the real dsDNA concentration.
  • Only 20uL 2nd MDA amplicon was purified and measured. Since the purification of whole 2nd MAD amplicon (200uL) is not effective and time consuming.


Samples

  • 200uL of 2nd MDA amplicons from:
    • 2ndMDA-L4N9(8hr), 2ndMDA-L4N9(4hr), 2ndMDA-L4N8(6hr), 2ndMDA-L4N8(4hr), 2ndMDA-L4N7(6hr), 2ndMDA-L4N7(4hr), 2ndMDA-N6(6hr) and 2ndMDA-N6(4hr)


Purification

  • Assemble the filter
  • Transfer 20uL of MDA amplcion into a 1.5-mL tube.
  • Add Nuclease-free H2O (Ambion) up to 200uL of total volume. Mix by vortexing for 10 sec and briefly spining down the mixture.
  • Transfer the mixture on the filter reservoir.
  • Cap the tube and centrifuge at 14,000xg for 12 min.
  • Add 20uL Nuclease-free H2O (Ambion) on the filter. Stay the filter for one min in RT. Invert and transfer the filter on a fresh collection tube. Centrifuge at 3000xg for 3 min.
  • Estimate the volume of elutant using tips. Nanodrop the dsDNA concentration.


Results

Results

 File:ZhangLab 2 2009-03-21 10hr 42min-annotate.jpg


Discussion

  • The kit purification protocol is working in this experiment. However, the effect of purification can't be confirmed through these results. More tests on p-pos samples may need to be done.
  • A1 - the MDA amplicon from gDNA tamplate covers 4 bands(genes) which can almost serves as a positive control.