Sam:LabNotes/Microbiome/2009-3-13: Difference between revisions

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='''Qiagen MPCR test with Q-solution on MDA L4N6/Tre amplicons'''=


==Objectives==
*Test of 20 uL volumn of QMPCR with Q-sol
*Confirm the amplicons from MDA L4N6/Tre
==Samples & Materials==
*Primers: '''Note: use the un-adjusted primer mixture'''
**Blue primers mix: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757)
*Templates: (Amplicon label form MDA reaction)
    Row A: B1 B2 B3 B4 B5 B7 B8
        B: C1 C2 C3 C8 A4 gDNA H2O
        Note: A4 = p-pos   
        Total= 14 rxns
*Master solution of 20 uL QMPCR with Q-sol.
                                    1 rxn      16 rxn
    -------------------------------------------------
    RNAse-free H2O                    5.0        80.0 
    Q-solution                        2.0        32.0
    Primer mix (Blue)                2.0        32.0
    Qiagen kit enzyme (2X)          10.0      160.0
    -------------------------------------------------
                                      19.0      304.0 (uL)
   
    Add 1 uL template
*QMPCR programs: QM-PCR52
      Step1  95C, 15m
      Step2  94C, 30s
      Step3  52C, 1m30s
      Step4  72C, 1m30s
      Step5  Goto Step2 X 30
      Step6  72C, 10m
      Step7  4C, forever
==Results==
[[Image:ZhangLab_2 2009-03-13 10hr 09min-with annotate.bmp|600px]]
*Gel Electrophoresis
**2 % agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe) -> two medium tray with 13-well comb
** Loading: Sample 10 uL + 2 uL loading buffer; Diluted ladder 10 uL
**Run at 135 V for 30 min.
==Discussion==
==Suggestion==

Latest revision as of 22:02, 16 March 2009