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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|600px]] *Gel Electrophoresis **2 % agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe) -> 4 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== *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 ==Suggestion== *Measure the DNA conc. by nonodrop for the template used in the experiment *Use the same amount DNA and same QMPCR condition (with Q-solution) to test all of the MDA amplicons again **Test with more different primer mix as well
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