Sam:LabNotes/Human/2009-4-12
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Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons
Procedure
- Experiment design
Primer 1-1->6-1 Primer 1-1->6-1 Column B6(3-31) B7(3-31)
- Thaw following regents:
- Taq pol.(2X)
- Primer Tamplate
- Primers (4 uL)
10 uL (10 mM of F + R) + 70 uL H2O mixture 1 rxn 20 rxn
Primer (f+r) 1.0 10 uL H2O 7.0 70 uL ------------------------------------ 8.0 80 uL Place diluted primer in eight PCR tubes(column 1 to 8 (left->right)) 1-1, 2-1, 3-1, 4-1, 1-1, 2-1, 3-1, 4-1
- Aliquots Taq2X enzymes into PCR tubes (100 uL) x 8 PCR tubes
- Preparing PCR strip tubes
- Using multi-channel pipette to upload all reagents
- Enzyme (5 uL): Taq Mix 2X
- Primer (4 uL)
- Using p2.5 pipettor to add diluted (1/50) Templates: (1 uL)
RowA (left) 3-31 B3, RowA(right) 3-31 B4 RowB (left) 3-31 B6, RowB(right) 3-31 C2 RowC (left) 3-31 C5, RowC(right) 3-31 C7 RowD (left) 3-31 C8, RowD(right) 3-31 gDNA 3 ng
- Program: 52T35C (Tm=52C, for 35 cycles
- Gel electrophoresis
- 2% agarose gel, midium tray with 26-well comb
- Sample (8uL) + Loading buffer (1.5 uL)
- Run at 135V for 20 min
Results
File:ZhangLab 2 2009-04-01 14hr 12min-annotate.jpg File:ZhangLab 2 2009-04-01 14hr 52min-annotate.jpg Primer(amplicon size) 1-1(270 bp), 2-1(540 bp), 3-1(235 bp), 4-1(383 bp)