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:

  1. Plate #1
    1. GM20431 cDNA
    2. GC1F cDNA
    3. GC1EP cDNA
  2. Plate #2
    1. Hues6 gDNA
    2. 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.