Sam:LabNotes/Microbiome/2009-3-11
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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
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