Sam:LabNotes/Microbiome/2009-3-11: Difference between revisions

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='''PCR confirmation for human MDA amplicons & Q-solution test on Qiagen MPCR kit'''=


==Objectives==
*Compare if the Q-solution can improve the results using Qiagen MPCR kit
*Pick up more candidates of amplicons to be tested using the Taq2X MPCR with set-up condition
==Samples & Materials==
*Primers:
**Blue primers mix: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757)
**Yello primers mix: 11-1(131), 10-1(214), 15-1(297), 17-1(419), 16-1(806)
*Templates
**For all Qiagen MPCR (QMPCR) tests: 5 uL of 1 ng/uL human gDNA
**For TaqPol2X MPCR (TMPCR) tests: 1 uL of Amplicon candidates picked up from human MDA (03/10/09): B10, E9, E10, F9, F10, H8, H9, H10
==Procedures==
*QMPCR - no Q-solution
                            1 rxn     
    -----------------------------
    RNAse-free H2O          15.0
    Template (1 ng/uL)        5.0 
    Qiagen kit enzyme (2X)  25.0
    -----------------------------
                              45.0 (uL)
    Add 5 uL primer mix Bule / Yellow (Use the unadjusted set -> equal ammount of each pair)
*QMPCR - with Q-solution
                            1 rxn
    -----------------------------
    RNAse-free H2O          10.0
    Q-solution                5.0
    Template (1 ng/uL)        5.0 
    Qiagen kit enzyme (2X)  25.0
    -----------------------------
                              45.0 (uL)
    Add 5 uL primer mix Bule / Yellow (Use the unadjusted set -> equal ammount of each pair)
*TMPCR '''Note: only 1 uL template was used since MDA amplicon have have very high concentration
                            1 rxn     
    -----------------------------
    RNAse-free H2O          19.0
    Primer Bule              5.0 (Use the djusted set)
    NEB Taq pol.(2X)        25.0
    -----------------------------
                              49.0 (uL)
    Add 1 uL MDA amplicon 
*QMPCR programs: QM-PCR52 '''Note: cycles were decreased from 35 to 30'''
      Step1  95C, 15m
      Step2  94C, 30s
      Step3  52C, 1m30s
      Step4  72C, 1m30s
      Step5  Goto Step2 X 30
      Step6  72C, 10m
      Step7  4C, forever
*QMPCR programs: TM-PCR52 '''Note: cycles were increaded from 35 to 40'''
      Step1  94C, 2m
      Step2  94C, 40s
      Step3  52C, 40s
      Step4  72C, 1ms
      Step5  Goto Step2 X 40
      Step6  72C, 10m
      Step7  4C, forever
==Results==
[[Image:ZhangLab_2 2009-03-12 10hr 09min-with annotate.bmp|800px]]
==Discussion==
*Adding Q-solution significantly increase the binding quality and can remove the extra band shown in previous results on both primer mix Blue and Yellow
*The testing of eight MDA candidates showed only one positive results B10

Latest revision as of 17:17, 18 March 2009