Template:SJTU-BioX-Shanghai/Notebook/Construction/w7

Construction

Aug. 17

1. Component cell making.

Aug. 20

1. Producted linearized Pdark-GFP, amplified GFP fragments from constructed plasmid GFP.
2. Agarose gel electrophoresis for PCR products.
3. Extracted positive plasmids (16 tubes).
4. Inoculated 14 D3 colonies and negative control C2/D2 for 2 tubes. (16 tubes in total)

Aug. 21

1. Extracted plasmids from the 16 tubes inoculated on 8.20.
2. Agarose gel electrophoresis test and restriction enzyme digestion test for plasmids above.(20μl system included 1μl SacI and 1μl EcoRI.)
3. Colony PCR test for D3 and C2/D2.
4. Prepared homologous recombination system (as is shown below).
5. Transformation and plating afterwards.

	GFP-1*	GFP-2*	GFP-3a	GFP-3b
ddwater	12μl	11μl	10μl	9μl
5x CEII Buffer	4μl	4μl	4μl	4μl
Linearized plasmid Pdark-HR(-GFP-)-1	3μl	3μl	3μl	3μl
GFP fragment	1μl	0μl	1μl	2μl
Exnase II	0μl(	2μl	2μl	2μl

• *GFP-1&2 are 2 control groups.

Aug. 22

Colony PCR for recombination cells. 12tubes for single colonies of GFP 3a and 3b, 3 tubes for control groups GFP-1 and GFP-2.

	GFP-3a/3b/1/2
ddwater	144μl
Template	0.5μl(each tube)
Primer M13F(10μM)	8μl
Primer GFP-R(10μM)	8μl
2xTaq Mix	160μl
Total volume	320μl(20μl*16)

As is shown in the figure, product no.6 showed negative and control no.1 showed positive. Most of the products qualified the length of 1400bp or so.

Aug. 23

Mass prep of Pdark-GFP plasmid (No.1,4).