Team:HokkaidoU Japan/Notebook/aggregation Week 9

Colony PCR

Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not.

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 695bp.

[[image:|thumb|Colony PCR result]]

Incubation for mini-prep of pT7-RBS-Ag43-dT on pSB1C3

Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.

1. Prepared 2 ml LBC into culture tubes.
2. Re-suspended 2 colonies (No.1 and No.2 respectively).
3. Incubated at 37C for 15 hrs.

Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2

1. Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Mixed 350 ul of LB.
4. Prepared and Labeled two plastic plates with LBC.
5. Plated 300 ul of the culture onto first dish and spread.
6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
7. Incubated the plates at 37C for 15 hours.

PCR of RBS-YFP-dT

Amplified the construct by 100bp-up-EX primer and 200bp-down-PS primer. Mixed PCR solutions.

Cycle:2~4 x 35

mini-prep of pT7-RBS-Ag43-dT on pSB1C3

Mini-prep of pT7-RBS-Ag43-dT on pSB1C3

[[image:|thumb|mini-prep result]]