IGEM:Peking/2007/Switch-Notebook/2007-7-10

Experimenter: LXL, LCB

Precipitate with ethanol: SDD-EGFP^^, pLX007^^

 * Time: 8:30

Colony PCR test of ECFP-pCC002

 * Primers: pkem-F, pCC-test-R



l to r: Marker, ECFP-pCC002(1-15), control

Select for incubation: ECFP-pCC002(6)

图像不清楚大约是因为Bobo错把TAE当了EB+TAE.

Transformation

 * SDD’-EGFP-pCC002
 * ECFP-pCC002
 * pCC002 (C/N)
 * CD1-pMD18T
 * RD1-pMD18T

Ligation

 * MCS-pCC009 4:1
 * pCC009 (self ligation control)

Temperature gradient PCR of RD2, RD3

 * Template: MG1655 FT Genome
 * Prime F: Rec_D_2_F/Rec_D_3_F
 * Prime R: Rec_S_2_R (new) / Rec_S_3_R (new)
 * Temperature: 50C, 53C, 58C, 63C, 68C



l to r: RD2(1-5), ladder

不浓, RD3也不浓. 问什么我P的就不浓呢? 呼唤NM师兄!

Ligation

 * pLX007, lacZa 1:4
 * pLX007 (x/s), self ligation control
 * pCC002-SDD-EGFP

ExTaq PCR

 * 35 cycles
 * Product: SS1, SS2, SS3, SD2 (X/S), SD3
 * Template: Psul-pMD18T, Psul-pMD18T, Psul-pMD18T, SD2-pMD18T, SD2-pMD18T
 * Primer_F: Sul_S_1_F, Sul_S_2_F, Sul_S_3_F, lacO2_XhoI_F, Sul_D_3_F
 * Primer_R: Sul_S_1_R, Sul_S_2_R, Sul_S_3_R, lacO2_XhoI_R, Sul_D_3_R



l to r: SS1, SS2, SS3, SD2 (X/S), SD3, ladder


 * Retract
 * Ligate SS1-3, SD3, pMD18T

要是SD2 XS P不出来就惨了, 我还得重写引物, 明天温度梯度试试吧.

Transformation

 * 7 Amp+: SS1-T, SS2-T, SS3-T, SD3-T, CD1-T, RD1-T, pLX007 (xs)自, pLX007-lacZa
 * Cm+: SDD_EGFP_pCC002