Kun:LabNotes/ASE/2007-10-16
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Exp.1: Repeat the PCR reactions on the PGP1VER2 primer set[edit]
cDNA primers on the plate
Forward primer | Reverse primer | Seq. Primer | |||||||||
A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | A11 | A12 |
B1 | B2 | B3 | B4 | B5 | B6 | B7 | B8 | B9 | B10 | B11 | B12 |
C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 | C9 | C10 | C11 | C12 |
D1 | D2 | D3 | D4 | D5 | D6 | D7 | D8 | D9 | D10 | D11 | D12 |
E1 | E2 | E3 | E4 | E5 | E6 | E7 | E8 | E9 | E10 | E11 | E12 |
F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 | F10 | F11 | F12 |
G1 | G2 | G3 | G4 | G5 | G6 | G7 | G8 | G9 | G10 | G11 | G12 |
H1 | H2 | H3 | H4 | H5 | H6 | H7 | H8 | H9 | H10 | H11 | H12 |
gDNA primers on the plate:
Forward primer | Reverse primer | Seq. Primer | |||||||||
A1 | A2 | A4 | A5 | A6 | A8 | A9 | A10 | A12 | |||
B1 | B4 | B5 | B8 | B9 | B12 | ||||||
C1 | C2 | C3 | C5 | C6 | C7 | C9 | C10 | C11 | |||
D2 | D3 | D6 | D7 | D10 | D11 | ||||||
E2 | E3 | E4 | E6 | E7 | E8 | E10 | E11 | E12 | |||
F1 | F3 | F4 | F5 | F7 | F8 | F9 | F11 | F12 | |||
G1 | G3 | G4 | G5 | G7 | G8 | G9 | G11 | G12 | |||
H1 | H2 | H3 | H5 | H6 | H7 | H9 | H10 | H11 |
I'm setting up 32 amplifications for each of the following templates:
- Plate #1
- GM20431 cDNA
- GC1F cDNA
- GC1EP cDNA
- Plate #2
- Hues6 gDNA
- Hues6 cDNA
Reactions:
One 33x Template 1ul 33ul 10X buffer 5ul 165ul 25mM MgCl2 3ul 99ul 10mM dNTP 1ul 33ul 50X SYBG I 0.4ul 13.2ul 10uM Primers 1ul each ThermoStart Taq 0.25ul 8.25ul H2O 38.35ul 1266ul
95C 15min -> 40 cycles of (94C 30S -> 57C 30S -> 72C 1min) -> 72C 3min on Chromo4.
Only no amplification worked for GM20431 cDNA, 2 worked for GC1F, about a dozen worked for GC1EP
Exp.2: Repeat Exp.1 on the three PGP1 cDNAs using NEB's 2X Taq Master mix[edit]
Set up reactions on ice:
One 33x Template 1ul 33ul 2X Taq Mix 25ul 825ul 50X SYBG I 0.4ul 13.2ul 10uM Primers 1ul each H2O 22.6ul 746ul
94C 2min -> 40 cycles of (94C 30S -> 56C 30S -> 68C 1min) -> 68C 3min on Chromo4.
Result[edit]
Few reactions with ThermoStart Taq worked. Taq 2x mix is a lot better, the curves for most amplification came up. I had some issues with running agarose gel. I check A1-A10 on 8% PAGE gel, and they look good. I will repeat the amplification on GM20431 gDNA, Hues6 gDNA, and Hues6 cDNA.