Team:HokkaidoU Japan/Notebook/aggregation Week 10
From 2012.igem.org
September 3rd
Colony PCR of eCFP-RBS-pSB1A2
DNA solution | 4 ul |
Kapa-Taq(Taq polymerase) | 5 ul |
Forward Primer(EX-F primer) | 0.5 ul |
Reverse Primer(PS-R down primer) | 0.5 ul |
Total | 10 ul |
Number | Degree | Second |
1 | 95 | 120 |
2 | 95 | 30 |
3 | 68.9 | 30 |
4 | 72 | 60 |
5 | 72 | 60 |
6 | 4 | HOLD |
Cycle:2~4 x 35
We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. Desired product is about 776bp.
We confirmed that about 70% of ligated DNA formed our desired construct. We selected No.2 and 5 colony for incubation and store No.7 and 8 colony mixture at 4C.
Incubation of eCFP-RBS-pSB1A2 for mini-prep
- Prepared 2 ml LBA into culture tubes.
- Re-suspended 2 colony mixture (No.2 and No.5 respectively).
- Incubated at 37C for hrs.
Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2
No.2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2. [[image:|thumb|electrophoresis result]] We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul and pBAD-RBS-pSB1A2 is 50ng/ul.
Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2
To make a construct of pBAD-RBS-eCFP-RBS-pSB1A2, we digested eCFP-RBS-pSB1A2 with XbaI & PstI and pBAD-RBS-pSB1A2 with SpeI & PstI. And we digested eCFP-pSB1A2 with XbaI & SpeI as a control for confirmation of the ability to digest. Insert (eCFP-RBS-pSB1A2)
DNA solution ( 30ng/ul) | 22 ul |
XbaI | 1 ul |
PstI | 1 ul |
10xM buffer | 3 ul |
DW | 3 ul |
Total | 30 ul |
Vector(pBAD-RBS-pSB1A2)
DNA solution ( 50ng/ul) | 3 ul |
SpeI | 1 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 13 ul |
Total | 20 ul |
control (eCFP-pSB1A2)
DNA solution (30 ng/ul) | 5 ul |
XbaI | 1 ul |
SpeI | 1 ul |
10xM buffer | 2 ul |
DW | 11 ul |
Total | 20 ul |
Number | Degree | Minute |
1 | 37 | 120 |
2 | 60 | 15 |
3 | 4 | HOLD |
[[image:|thumb|digestion result]]
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked.
Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged in 15000 rpm, 15 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged in 15000 rpm, 10 min at 4C.
- Remove supernatant and air drying in room temperature then added 5 ul of DW.
Ligation of eCFP-RBS and pBAD-RBS-pSB1A2
Vector DNA (50 ng/ul) | 1.5 ul |
Insert DNA (20 ng/ul) | 4 ul |
Ligation Mighty Mix | 6 ul |
DW | 0.5 ul |
Total | 12 ul |
Ligation reaction time was in detail below.
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
Transformation of pBAD-RBS-eCFP-RBS-pSB1A2
- Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Mixed 350 ul of LB.
- Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
- Plated 300 ul of the culture onto first dish and spread.
- Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
- Incubated the plates at 37C for hours.